Process for the preparation of oxidized phospholipids

ABSTRACT

Novel synthetic routes, which are highly applicable for industrial preparation of therapeutically beneficial oxidized phospholipids are disclosed. Particularly, novel methods for efficiently preparing compounds having a glycerolic backbone and one or more oxidized moieties attached to the glycerolic backbone, which are devoid of column chromatography are disclosed. Further disclosed are novel methods of introducing phosphorous-containing moieties such as phosphate moieties to compounds having glycerolic backbone and intermediates formed thereby.

RELATED APPLICATIONS

This is application is a divisional of U.S. patent application Ser. No.11/650,973, filed on Jan. 9, 2007, which is a continuation-in-part ofInternational Patent Application No. PCT/IL2005/000735 (WO2006/006161),filed on Jul. 10, 2005, which claims the benefit of priority from U.S.Provisional Patent Application No. 60/586,219, filed on Jul. 9, 2004,the teachings of both being incorporated herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the field of synthetic chemistry, andmore particularly, to novel synthetic processes useful for thepreparation of oxidized phospholipids, derivatives, analogs and saltsthereof.

In the art of pharmacology, modified phospholipids are known in manyapplications. In U.S. Pat. No. 5,985,292 compositions for trans-dermaland trans-membranal application incorporating phospholipids bearinglipid-soluble active compounds are disclosed. In U.S. Pat. Nos.6,261,597, 6,017,513 and 4,614,796 phospholipid derivatives incorporatedinto liposomes and biovectors for drug delivery are disclosed. In U.S.Pat. No. 5,660,855 lipid constructs of aminomannose-derivatizedcholesterol suitable for targeting smooth muscle cells or tissue,formulated in liposomes, are disclosed. These formulations are aimed atreducing restenosis in arteries, using PTCA procedures.

The use of liposomes for treating atherosclerosis has been furtherdisclosed in the PCT patent application published as WO 95/23592.Therein are disclosed pharmaceutical compositions of unilamellarliposomes that may contain phospholipids. The liposomes disclosed in WO95/23592 are aimed at optimizing cholesterol efflux from atheroscleroticplaque and are typically non-oxidized phospholipids.

Modified phospholipid derivatives mimicking platelet activation factor(PAF) structures are known to be pharmaceutically active, affecting suchfunctions as vascular permeability, blood pressure and heart functioninhibition. In U.S. Pat. No. 4,778,912 it is suggested that one group ofsuch derivatives has anti-cancer activity.

In U.S. Pat. No. 4,329,302 synthetic 1-O-alkyl ether or 1-O-fatty acylphosphoglycerides compounds which are lysolechitin derivatives usable inmediating platelet activation are disclosed. In U.S. Pat. No. 4,329,302is disclosed that small chain acylation of lysolechitin gave rise tocompounds with platelet activating behavior, as opposed to long-chainacylation, and that the 1-O-alkyl ether are biologically superior to thecorresponding 1-O-fatty acyl derivatives in mimicking PAF.

The structural effect of various phospholipids on the biologicalactivity thereof has been investigated by Tokumura et al. (Journal ofPharmacology and Experimental Therapeutics. July 1981, Vol. 219, No. 1)and in U.S. Pat. No. 4,827,011, with respect to hypertension.

In Swiss patent CH 642,665 modified phospholipid ether derivatives thatmay have some physiological effect are disclosed.

Davies et al. (J. Biol. Chem. 2001, 276:16015) teach the use of oxidizedphospholipids as peroxisome proliferator-activated receptor agonists.

In U.S. Pat. No. 6,838,452 and in WO 04/106486 (which are incorporatedby reference as if fully set forth herein), by the present assignee, thepreparation of well-defined oxidized phospholipids, as well as othersynthetic oxidized LDL (low density lipoprotein) components, isdisclosed. The disclosed compounds are reported to be highly effectivein treating atherosclerosis and related diseases, as well as autoimmunediseases and inflammatory disorders. It is further reported that theoxidized phospholipid regulate the immune response to oxidized LDL. Itis further reported that generally, etherified oxidized phospholipidsare superior to comparable esterified oxidized phospholipids astherapeutic agents.

Oxidation of phospholipids occurs in vivo through the action of freeradicals and enzymatic reactions abundant in atheromatous plaque. Invitro, preparation of oxidized phospholipids usually involves simplechemical oxidation of a native LDL or LDL phospholipid component.Investigators studying the role of oxidized LDL have employed, forexample, ferrous ions and ascorbic acid (Itabe, H., et al., J. Biol.Chem. 1996; 271:33208-217) and copper sulfate (George, J. et al.,Atherosclerosis. 1998; 138:147-152; Ameli, S. et al., ArteriosclerosisThromb Vasc Biol 1996; 16:1074-79) to produce oxidized, or mildlyoxidized phospholipid molecules similar to those associated with plaquecomponents. Similarly prepared molecules have been shown to be identicalto auto-antigens associated with atherogenesis (Watson A. D. et al., J.Biol. Chem. 1997; 272:13597-607) and able to induce protectiveanti-atherogenic immune tolerance (U.S. patent application Ser. No.09/806,400 to Shoenfeld et al., filed Sep. 30, 1999) in mice. Similarly,in U.S. Pat. No. 5,561,052, a method of producing oxidized lipids andphospholipids using copper sulfate and superoxide dismutase to produceoxidized arachidonic or linoleic acids and oxidized LDL for diagnosticuse is disclosed.

The oxidation techniques described above for preparing oxidizedphospholipids involve reactions that are non-specific and yield amixture of oxidized products. The non-specificity of the reactionsreduces yield, requires a further separation step and raises concern forundesired side effects when the products are integrated inpharmaceutical compositions.

1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) andderivatives thereof such as1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine GPC) arerepresentative examples of mildly oxidized esterified phospholipids thathave been studied with respect to atherogenesis (see, for example,Boullier et al., J. Biol. Chem. 2000, 275:9163; Subbanagounder et al.,Circulation Research, 1999, pp. 311). The effect of different structuralanalogs that belong to this class of oxidized phospholipids has alsobeen studied (see, for example, Subbanagounder et al., Arterioscler.Thromb. Nasc. Biol. 2000, pp. 2248; Leitinger et al., Proc. Nat. Ac.Sci. 1999, 96:12010).

POVPC is typically prepared by providing a phosphatidyl choline bearingan unsaturated fatty acid and oxidizing the unsaturated bond of thefatty acid by, e.g., ozonolysis (oxidative cleavage) or using aperiodate as an oxidizing agent. Such a synthetic pathway typicallyinvolves a multi-step synthesis and requires separation of most of theformed intermediates by means of column chromatography.

As described in U.S. Pat. No. 6,838,452 cited above, etherified oxidizedphospholipids have been similarly prepared by oxidizing an unsaturatedbond of a fatty acid attached to a phospholipid backbone. Moreparticularly, the etherified oxidized phospholipids were prepared,according to the teachings of this patent, by introducing an unsaturatedshort fatty acid to a glycerolipid, introducing a phosphate moiety tothe obtained intermediate and oxidizing the unsaturated bond in thefatty acid chain by means of (i) hydrogen peroxide and formic acid, soas to obtain a diol, followed by potassium periodate, so as to obtain analdehyde; or (ii) ozonolysis. While the oxidative cleavage of theunsaturated bond results in an aldehyde moiety, other oxidized moieties(e.g., carboxylic acid, acetal, etc.) were obtained by further oxidizingthe aldehyde moiety. Such a multi-step synthetic pathway is oftentimescharacterized by relatively low overall yields and again, requiresseparation of most of the formed intermediates by means of columnchromatography.

It has been found that in vivo applications employing esterifiedoxidized phospholipids prepared as above have the disadvantage ofsusceptibility to recognition, binding and metabolism of the activecomponent in the body, making dosage and stability after administrationan important consideration. Etherified oxidized phospholipids, such asthose described in U.S. Pat. No. 6,838,452 and in WO 04/106486, exhibithigher biostability and high therapeutic activity.

Thus, the currently known methods of preparing etherified, as well asesterified, oxidized phospholipids involve complex multi-step proceduressuitable for laboratory preparation yet rendering industrial scalepreparation inefficient and complex. In particular, these multi-stepprocedures require industrially inapplicable separation techniques suchas column chromatography during various stages of the synthetic process.

In view of the beneficial therapeutic activity of oxidized phospholipidsin general and of etherified oxidized phospholipids in particular, thereis a widely recognized need for and it would be highly advantageous tohave an improved process for the preparation of etherified oxidizedphospholipids devoid of at least some of the disadvantages of processesknown in the art.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of preparing a compound having a glycerolic backbone and at leastone oxidized moiety-containing residue attached to the glycerolicbackbone via an ether bond, which comprises: providing a first compoundhaving a glycerolic backbone and at least one free hydroxyl group;providing a second compound having at least one unsaturated bond and atleast one reactive group capable of forming an ether bond with the freehydroxyl group; reacting the first compound and the second compound tothereby obtain a third compound, the third compound having a glycerolicbackbone and an unsaturated bond-containing residue being attached tothe glycerolic backbone via an ether bond; isolating the third compound,to thereby obtain a purified third compound; reacting the purified thirdcompound with an oxidizing agent, to thereby obtain a fourth compound,the fourth compound having a glycerolic backbone and an oxidizedmoiety-containing residue attached to the glycerolic backbone via anether bond; and isolating the fourth compound to thereby obtain apurified fourth compound, thereby obtaining the compound having aglycerolic backbone and at least one oxidized moiety-containing residueattached to the glycerolic backbone via an ether bond, the method beingdevoid of column chromatography.

According to further features in preferred embodiments of the inventiondescribed below, reacting the first compound and the second compound iscarried out in the presence of a base.

According to still further features in the described preferredembodiments the base is selected from the group consisting of sodiumhydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide andpotassium hydroxide.

According to still further features in the described preferredembodiments the reactive group is a halide.

According to still further features in the described preferredembodiments isolating the third compound comprises: collecting the thirdcompound; providing a solution of the third compound in a solvent, thesolvent being selected such that the third compound is soluble thereinwhereby impurities formed during the reacting are insoluble therein, tothereby provide a mixture including the solution of the third compoundin the solvent and insoluble impurities; removing the insolubleimpurities; and removing the solvent, thereby obtaining the purifiedthird compound.

According to still further features in the described preferredembodiments the solvent is selected from the group consisting of petrolether, hexane and benzene.

According to still further features in the described preferredembodiments the oxidizing agent is selected from the group consisting offormic acid, hydrogen peroxide, a periodate, a perchlorate, abismuthate, a permanganate, a chlorite, ozone, silver oxide, osmiumtetraoxide and any combination thereof.

According to still further features in the described preferredembodiments the oxidized moiety is selected from the group consisting ofa carboxylic acid, an ester, an aldehyde, an acetal, a ketal and a diol.

According to still further features in the described preferredembodiments the oxidized moiety is aldehyde and reacting the purifiedthird compound with the oxidizing agent comprises: converting thepurified third compound to a compound having a glycerolic backbone and adiol-containing residue attached to the glycerolic backbone via an etherbond; and oxidizing the compound having a glycerolic backbone and adiol-containing residue attached to the glycerolic backbone, to therebyobtain the fourth compound having a glycerolic backbone and analdehyde-containing residue attached to the glycerolic backbone via anether bond.

According to still further features in the described preferredembodiments the converting is effected by reacting the purified thirdcompound with a first oxidizing agent selected from the group consistingof a peroxide, a bismuthate, a periodate, a permanganate, and anycombination thereof.

According to still further features in the described preferredembodiments the oxidizing is effected by reacting the compound having aglycerolic backbone and a diol-containing residue attached to theglycerolic backbone with a second oxidizing agent selected from thegroup consisting of a periodate, a bismuthate, a permanganate, and achlorite

According to still further features in the described preferredembodiments isolating the fourth compound comprises: collecting thefourth compound; providing a water-soluble adduct of the fourthcompound; subjecting the water-soluble adduct to a biphasic system, tothereby provide an aqueous phase containing the adduct and an organicphase containing water-insoluble impurities formed during the reactingwith the oxidizing agent; collecting the aqueous phase; decomposing theadduct; and collecting the fourth compound, thereby obtaining thepurified fourth compound.

According to still further features in the described preferredembodiments providing the water-soluble adduct comprises: reacting thefourth compound with a Girard reagent.

According to still further features in the described preferredembodiments the oxidized moiety is a carboxylic acid and reacting thepurified third compound with the oxidizing agent comprises: convertingthe purified third compound to a compound having a glycerolic backboneand an aldehyde-containing residue attached to the glycerolic backbonevia an ether bond; and oxidizing the compound having a glycerolicbackbone and an aldehyde-containing residue attached to the glycerolicbackbone, to thereby obtain a compound having a glycerolic backbone anda carboxylic acid-containing residue attached to the glycerolic backbonevia an ether bond.

According to still further features in the described preferredembodiments converting the purified third compound to the compoundhaving a glycerolic backbone and an aldehyde-containing residue attachedto the glycerolic backbone via an ether bond comprises: converting thepurified third compound to a compound having a glycerolic backbone and adiol-containing residue attached to the glycerolic backbone via an etherbond; and oxidizing the compound having a glycerolic backbone and adiol-containing residue attached to the glycerolic backbone, to therebyobtain the compound having a glycerolic backbone and analdehyde-containing residue attached to the glycerolic backbone via anether bond.

According to still further features in the described preferredembodiments the method further comprises isolating the compound having aglycerolic backbone and an aldehyde-containing residue attached to theglycerolic backbone via an ether bond, to thereby obtain a purifiedcompound having a glycerolic backbone and an aldehyde-containing residueattached to the glycerolic backbone via an ether bond.

According to still further features in the described preferredembodiments the isolating comprises: collecting the compound having aglycerolic backbone and an aldehyde-containing residue attached to theglycerolic backbone via an ether bond; providing a water-soluble adductof the compound having a glycerolic backbone and an aldehyde-containingresidue attached to the glycerolic backbone via an ether bond, asdescribed hereinabove; subjecting the water-soluble adduct to a biphasicsystem, to thereby provide an aqueous phase containing the complex andan organic phase containing water-insoluble impurities formed during theconverting and/or the oxidizing; collecting the aqueous phase;decomposing the adduct; and collecting the compound having a glycerolicbackbone and an aldehyde-containing residue attached to the glycerolicbackbone via an ether bond, thereby obtaining a purified compound havinga glycerolic backbone and an aldehyde-containing residue attached to theglycerolic backbone via an ether bond.

According to still further features in the described preferredembodiments the oxidized moiety is a carboxylic acid and reacting thepurified third compound with the oxidizing agent comprises: convertingthe purified third compound to a compound having a glycerolic backboneand an epoxide-containing residue attached to the glycerolic backbonevia an ether bond; and oxidizing the compound having a glycerolicbackbone and an epoxide-containing residue attached to the glycerolicbackbone, to thereby obtain a compound having a glycerolic backbone anda carboxylic acid-containing residue attached to the glycerolic backbonevia an ether bond.

According to still further features in the described preferredembodiments the converting comprises reacting the third compound with aperoxide.

According to still further features in the described preferredembodiments the oxidized moiety is a carboxylic acid and reacting thepurified third compound with the oxidizing agent comprises reacting thepurified third compound with a mixture of a permanganate and aperiodate.

According to still further features in the described preferredembodiments the said reacting is effected in the presence of a base.

According to still further features in the described preferredembodiments the first compound has at least two free hydroxyl groups,the method further comprising, prior to the reacting the first compoundand the second compound: protecting at least one of the at least twogroups with a protecting group.

According to still further features in the described preferredembodiments the protecting group is trityl.

According to still further features in the described preferredembodiments the first compound has at least two free hydroxyl groups,the method further comprising, prior to the reacting the first compoundand the second compound: protecting at least one of the at least twogroups with a protecting group, preferably a trityl group.

According to still further features in the described preferredembodiments, when the methods include the formation of anepoxide-containing compound, as described hereinabove, the methodfurther comprises, prior to reacting the third compound and theoxidizing agent: replacing the trityl with a protecting group selectedfrom the group consisting of acetate, pivaloate or benzoate.

According to still further features in the described preferredembodiments the compound having a glycerolic backbone and at least oneoxidized moiety-containing residue attached to the glycerolic backbonefurther comprises a phosphorous-containing moiety attached to theglycerolic backbone, and the method further comprises, prior to reactingthe first compound and the second compound, prior to isolating the thirdcompound, prior to reacting the third compound with the oxidizing agent,prior to isolating the fourth compound or subsequent to isolating thefourth compound:

reacting the first compound, the third compound, the purified thirdcompound, the fourth compound or the purified fourth compound with aphosphorous-containing moiety, to thereby obtain the compound having aglycerolic backbone and at least one oxidized moiety-containing residueattached to the glycerolic backbone and further having aphosphorous-containing moiety attached to the glycerolic backbone.

According to still further features in the described preferredembodiments the at least one phosphorous-containing moiety is aphosphate moiety being attached to the glycerolic backbone via aphosphodiester bond.

According to still further features in the described preferredembodiments the at least one phosphorous-containing moiety is selectedfrom the group consisting of phosphoric acid, phosphoryl choline,phosphoryl ethanolamine, phosphoryl serine, phosphoryl cardiolipin,phosphoryl inositol, ethylphosphocholine, phosphorylmethanol,phosphorylethanol, phosphorylpropanol, phosphorylbutanol,phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine and phosphoglycerol.

According to still further features in the described preferredembodiments the phosphorous-containing moiety is attached to the sn-3position of the glycerolic backbone of the compound.

According to still further features in the described preferredembodiments reacting the first compound, the third compound, thepurified third compound, the fourth compound or the purified fourthcompound with the phosphorous-containing moiety comprises: providing thefirst compound, the third compound, the purified third compound, thefourth compound or the purified fourth compound having a free hydroxylgroup; reacting the first compound, the third compound, the purifiedthird compound, the fourth compound or the purified fourth compound witha reactive phosphorous-containing compound having a second reactivegroup and a third reactive group, the second reactive group beingcapable of reacting with the free hydroxyl group and a second reactivegroup, to thereby provide the first compound, the third compound, thepurified third compound, the fourth compound or the purified fourthcompound having a reactive phosphorous-containing group attached to theglycerolic backbone; and converting the reactive phosphorous-containinggroup to the phosphorous-containing moiety.

According to still further features in the described preferredembodiments the reactive phosphorous-containing compound is phosphorousoxychloride (POCl₃).

According to still further features in the described preferredembodiments the reacting is carried out in the presence of a base.

According to still further features in the described preferredembodiments the phosphorous-containing moiety is phosphoric acid, andthe converting comprises hydrolyzing the reactive phosophrous-containinggroup.

According to still further features in the described preferredembodiments the phosphorous-containing moiety comprises an aminoalkylgroup and the converting comprises reacting the reactivephosophrous-containing group with a derivative of the aminoalkyl group,the derivative being selected capable of reacting with the thirdreactive group.

According to another aspect of the present invention there is providedanother method of preparing a compound having a glycerolic backbone andat least one oxidized moiety-containing residue attached to theglycerolic backbone via an ether bond, the method comprising: providinga first compound having a glycerolic backbone and at least one freehydroxyl group; providing a fifth compound having at least one oxidizedmoiety and at least one fourth reactive group; reacting the firstcompound and the fifth compound to thereby obtain a reaction mixturecontaining a sixth compound, the sixth compound being the compoundhaving a glycerolic backbone and at least one oxidized moiety-containingresidue attached to the glycerolic backbone via an ether bond; andisolating the compound having a glycerolic backbone and at least oneoxidized moiety-containing residue attached to the glycerolic backbonevia an ether bond.

According to further features in preferred embodiments of the inventiondescribed below, reacting the first compound and the fifth compound iseffected in the presence of a base.

According to still further features in the described preferredembodiments the base is selected from the group consisting of sodiumhydride, lithium aluminum hydride, sodium amide, sodium hydroxide,potassium hydroxide and any mixture thereof.

According to still further features in the described preferredembodiments the fourth reactive group is a halide.

According to still further features in the described preferredembodiments the oxidized moiety is selected from the group consisting ofa carboxylic acid, an ester, an acyl halide, an aldehyde, an acetal, aketal and a diol

According to still further features in the described preferredembodiments the fifth compound comprises less than 4 carbon atoms.

According to still further features in the described preferredembodiments the fifth compound comprises more than 5 carbon atoms.

According to still further features in the described preferredembodiments the first compound has at least two free hydroxyl groups,the method further comprising, prior to the reacting the first compoundand the fifth compound: protecting at least one of the at least twogroups with a protecting group.

According to still further features in the described preferredembodiments the protecting group is trityl.

According to still further features in the described preferredembodiments the compound having a glycerolic backbone and at least oneoxidized moiety-containing residue attached to the glycerolic backbonefurther comprises a phosphorous-containing moiety attached to theglycerolic backbone, the method further comprising, prior to orsubsequent to reacting the first compound and the fifth compound, orsubsequent to isolating the sixth compound: reacting the first compoundor the sixth compound with a phosphorous-containing moiety, to therebyobtain the compound having a glycerolic backbone and at least oneoxidized moiety-containing residue attached to the glycerolic backboneand further having a phosphorous-containing moiety attached to theglycerolic backbone, as described hereinabove.

According to further features in preferred embodiments of the inventiondescribed below, in any of the methods described herein, the firstcompound further comprises at least one alkylene chain having 1-30carbon atoms.

According to still further features in the described preferredembodiments the alkylene chain is attached to the glycerolic backbonevia an ether bond.

According to still further features in the described preferredembodiments the alkylene chain is attached to the sn-1 position of theglycerolic backbone of the first compound.

According to still further features in the described preferredembodiment the oxidized moiety-containing residue is attached to thesn-2 position of the compound and further wherein at least one of thefree hydroxyl groups of the glycerolic backbone is at the sn-2 positionof the first compound.

According to still further features in the described preferredembodiments the first compound has the general formula I:

wherein:

A₁ is absent or is selected from the group consisting of CH₂, CH═CH andC═O;

R₁ is selected from the group consisting of H and a hydrocarbon chainhaving from 1 to 30 carbon atoms; and

R₃ is selected from the group consisting of hydrogen, alkyl, aryl,phosphoric acid, phosphoryl choline, phosphoryl ethanolamine, phosphorylserine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylserine, phosphatidyl cardiolipin, phosphatidyl inositol, phosphorylcardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, phosphoglycerol.

According to still further features in the described preferredembodiments the compound having a glycerolic compound and at least oneoxidized moiety attached to the glycerolic backbone via an ether bondhas the general Formula II:

wherein:

A₁ is selected from the group consisting of CH₂, CH═CH and C═O;

A₂ is CH₂;

R₁ is an alkyl having 1-30 carbon atoms;

R₂ is

whereas:

X is an alkyl chain having 1-24 carbon atoms;

Y is selected from the group consisting of hydrogen, hydroxy, alkyl,alkoxy, halide, acetoxy and an aromatic functional group; and

Z is selected from the group consisting of:

with R₄ being an alkyl or aryl; and

R₃ is selected from the group consisting of hydrogen, alkyl, aryl,phosphoric acid, phosphoryl choline, phosphoryl ethanolamine, phosphorylserine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylserine, phosphatidyl cardiolipin, phosphatidyl inositol, phosphorylcardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, phosphoglycerol.

According to still another aspect of the present invention there isprovided a method of introducing a phosphate moiety into a compoundhaving a glycerolic backbone and having an oxidized moiety-containing ora pre-oxidized moiety-containing residue attached thereto via an etherbond, which comprises: providing a compound having a glycerolic backboneand an oxidized moiety- or a pre-oxidized moiety-containing residueattached to the glycerolic backbone via an ether bond and at least one afree hydroxyl group; reacting the compound a phosphorous-containingcompound having a second reactive group and a third reactive, the secondreactive group being capable of reacting with the free hydroxyl group,to thereby provide a compound having an oxidized moiety- or apre-oxidized moiety-containing residue and a reactivephosphorous-containing group; and converting the reactivephosphorous-containing group to the phosphate moiety, therebyintroducing the phosphate moiety into the compound.

According to further features in preferred embodiments of the inventiondescribed below, the compound having the glycerolic backbone comprisesat least one alkylene chain having 1-30 carbon atoms.

According to still further features in the described preferredembodiments the alkylene chain is attached to the glycerolic backbonevia an ether bond.

According to still further features in the described preferredembodiments the alkylene chain is attached to the sn-1 position of theglycerolic backbone of the compound.

According to still further features in the described preferredembodiments the oxidized moiety is selected from the group consisting ofcarboxylic acid, ester, acyl halide, aldehyde, acetal, diol and ketal.

According to still further features in the described preferredembodiments the pre-oxidized moiety is an unsaturated moiety.

According to still further features in the described preferredembodiments the phosphorous-containing compound is POCl₃.

According to still further features in the described preferredembodiments the reacting is performed in the presence of a base.

According to still further features in the described preferredembodiments the base is a tertiary amine.

According to still further features in the described preferredembodiments the phosphorous-containing compound is POCl₃, and thereactive phosphorous-containing group is a dichlorophosphate group.

According to still further features in the described preferredembodiments the compound having the glycerolic backbone has apre-oxidized moiety-containing residue attached thereto via an etherbond.

According to still further features in the described preferredembodiments the phosphate moiety is selected from the group consistingof phosphoric acid, phosphoryl choline, phosphoryl ethanolamine,phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,phosphoryl cardiolipin, ethylphosphocholine, phosphorylmethanol,phosphorylethanol, phosphorylpropanol, phosphorylbutanol,phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, phosphoglycerol.

According to still further features in the described preferredembodiments the phosphate moiety is phosphoric acid and the convertingcomprises hydrolyzing the reactive phosphorous-containing group.

According to still further features in the described preferredembodiments the phosphate moiety comprises an alkylamino group and theconverting comprises reacting the reactive phosphorous-containing moietywith a derivative of an aminoalkyl, the derivative being capable ofreacting with the reactive phosphorous-containing group.

According to still another aspect of the present invention there isprovided a method of preparing a compound having a glycerolic compoundand at least one oxidized moiety attached to the glycerolic backbone viaan ether bond, the compound having the general Formula II:

wherein: A₁ is selected from the group consisting of CH₂, CH═CH and C═O;A₂ is CH₂; R₁ is an alkyl having 1-30 carbon atoms; R₂ is

whereas: X is an alkyl chain having 1-24 carbon atoms; Y is selectedfrom the group consisting of hydrogen, hydroxy, alkyl, alkoxy, halide,acetoxy and an aromatic functional group; and Z is selected from thegroup consisting of:

with R₄ being an alkyl or aryl; and R₃ is selected from the groupconsisting of hydrogen, alkyl, aryl, phosphoric acid, phosphorylcholine, phosphoryl ethanolamine, phosphoryl serine, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidylcardiolipin, phosphatidyl inositol, phosphoryl cardiolipin, phosphorylinositol, ethylphosphocholine, phosphorylmethanol, phosphorylethanol,phosphorylpropanol, phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N4-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, phosphoglycerol, the methodcomprising:

providing a first compound having a glycerolic backbone and at least onefree hydroxyl group, the first compound having general Formula I:

providing a second compound having at least one unsaturated bond and atleast one reactive group capable of forming an ether bond with the freehydroxyl group; reacting the first compound and the second compound tothereby obtain a third compound, the third compound having theglycerolic backbone and an unsaturated bond-containing residue beingattached to the glycerolic backbone via an ether bond at position sn-2;isolating the third compound, to thereby obtain a purified thirdcompound; reacting the purified third compound with an oxidizing agent,to thereby obtain a fourth compound, the fourth compound having theglycerolic backbone and an oxidized moiety-containing residue attachedto the glycerolic backbone via an ether bond at position sn-2; andisolating the fourth compound to thereby obtain a purified fourthcompound, thereby obtaining the compound having a glycerolic backboneand at least one oxidized moiety-containing residue attached to theglycerolic backbone via an ether bond, the method being devoid of columnchromatography.

According to further features in preferred embodiments of the inventiondescribed below, isolating the third compound comprises: collecting thethird compound; providing a solution of the third compound in a solvent,the solvent being selected such that the third compound is solubletherein whereby impurities formed during the reacting are insolubletherein, to thereby provide a mixture including the solution of thethird compound in the solvent and insoluble impurities; removing theinsoluble impurities; and removing the solvent, thereby obtaining thepurified third compound.

According to still further features in the described preferredembodiments the oxidized moiety is selected from the group consisting ofa carboxylic acid and an ester.

According to still further features in the described preferredembodiments the oxidizing agent comprises a mixture of a periodate and apermanganate.

According to still further features in the described preferredembodiments reacting the purified third compound with an oxidizing agentis effected in the presence of a base.

According to still further features in the described preferredembodiments wherein R₃ is hydrogen, the method further comprising, priorto the reacting the first compound and the second compound: protecting afree hydroxyl group at position sn-3 of the glycerolic backbone with aprotecting group.

According to still further features in the described preferredembodiments the compound having a glycerolic backbone and at least oneoxidized moiety-containing residue attached to the glycerolic backbonefurther comprises a phosphorous-containing moiety attached to theglycerolic backbone, such that R₃ is selected from the group consistingof phosphoric acid, phosphoryl choline, phosphoryl ethanolamine,phosphoryl serine, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl serine, phosphatidyl cardiolipin, phosphatidyl inositol,phosphoryl cardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, phosphoglycerol, the method furthercomprising, subsequent to isolating the fourth compound: reacting thepurified fourth compound with a phosphorous-containing moiety, tothereby obtain the compound having a glycerolic backbone and at leastone oxidized moiety-containing residue attached to the glycerolicbackbone and further having a phosphorous-containing moiety attached tothe glycerolic backbone.

According to still further features in the described preferredembodiments the at least one phosphorous-containing moiety is aphosphate moiety being attached to the glycerolic backbone via aphosphodiester bond.

According to still further features in the described preferredembodiments reacting the purified fourth compound with thephosphorous-containing moiety comprises: providing the purified fourthcompound having a free hydroxyl group; reacting the purified fourthcompound with a reactive phosphorous-containing compound having a secondreactive group and a third reactive group, the second reactive groupbeing capable of reacting with the free hydroxyl group and a secondreactive group, to thereby provide the first compound, the thirdcompound, the purified third compound, the fourth compound or thepurified fourth compound having a reactive phosphorous-containing groupattached to the glycerolic backbone; and converting the reactivephosphorous-containing group to the phosphorous-containing moiety.

According to still further features in the described preferredembodiments the reactive phosphorous-containing compound is phosphorousoxychloride (POCl₃).

The present invention successfully addresses the shortcomings of thepresently known configurations by providing novel synthetic routes thatcan be beneficially used in the scaled-up preparation of oxidizedphospholipids.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein the term “mixture” describes a mixture that includes morethan one substance and which can be in any form, for example, as ahomogenous solution, a suspension, a dispersion, a biphasic solution andmore.

As used in this application, the singular form “a”, “an” and “the”include plural references unless the context clearly dictates otherwise.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein throughout, the terms “comprising”, “including” and“containing” means that other steps and ingredients that do not affectthe final result can be added. These terms encompass the terms“consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

The term “method” or “process” refers to manners, means, techniques andprocedures for accomplishing a given task including, but not limited to,those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

The term “phospholipid” is used herein to collectively describecompounds that include a non-polar lipid group and a highly polar endphosphate group. One particular and most prevalent in nature family ofphospholipid compounds is the phosphoglycerides family of compounds. Theterm “phospholipid” is therefore typically used herein throughout todescribe phosphoglycerides, unless otherwise indicated.

The term “phosphoglyceride” is therefore used herein to describecompounds having a glycerol backbone, one or more lipid moieties and oneor more phosphate end group, which are attached to the glycerolicbackbone. Most of the naturally-occurring glycerolipids include twolipid moieties attached to the sn-1 and sn-2 positions and one phosphatemoiety attached to the sn-3 position of the glycerol backbone.

The term “oxidized phospholipid” is therefore used herein to describe aphospholipid, as well as a phosphoglyceride, which includes one or moreoxidized moieties, as this term is described hereinbelow. Typically, inoxidized phospholipids, the oxidized moiety is included within a lipidmoiety.

The term “glycerolipid” describes a compound having a glycerolicbackbone and one or two lipid moieties attached thereto. The lipidmoieties can be attached to the glycerol backbone via an ester and/or anether bond.

As used herein, the term “lipid” describes a hydrocarbon residue having3-30 carbon atoms. In naturally-occurring compounds, the lipids inphospholipids and glycerolipids are derived from fatty acids and aretherefore attached to the backbone via an O-acyl(ester) bond. Herein,the lipid moiety can be attached to the backbone either via and ether oran ester bond.

As used herein, the terms “mono-esterified” and “di-esterified” withrespect to phospholipids or glycerolipids, describe phospholipids orglycerolipids, either oxidized or non-oxidized, in which one or two ofthe lipid moieties, respectively, are attached to the glycerol backbonevia an ester (e.g., O-fatty acyl) bond.

As used herein, the terms “mono-etherified” and “di-etherified” withrespect to phospholipids or glycerolipids, describe phospholipids orglycerolipids, either oxidized or non-oxidized, in which one or two ofthe lipid moieties, respectively, are attached to the glycerol backbonevia an ether bond.

The term “phosphoglycerol” describes a compound having a glycerolicbackbone and a phosphate group attached to one position thereof.

The term “phosphoglycerides” describes a compound having a glycerolicbackbone, one or two lipid moieties and a phosphate moiety attachedthereto.

The term “mono-etherified phosphoglyceride” describes aphosphoglyceride, in which a lipid moiety is attached to the glycerolicbackbone via an ether bond.

As used herein, the term “moiety” describes a functional substance orgroup which forms a part of a compound.

The term “residue” as is well known in the art, is used to described amajor portion of a molecule that is linked to another molecule.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of novel methods of preparing oxidizedphospholipids which can be efficiently used for a scaled up productionof such oxidized phospholipids. Specifically, the present invention isof novel methods of introducing an oxidized moiety to a compound havinga glycerolic backbone and is further of novel methods of introducing aphosphorous-containing moiety to such a compound. The novel methodsdescribed herein are devoid of column chromatography and typically usecommercially available and environmental friendly reactants.

The principles and operation of the novel synthetic methods according tothe present invention may be better understood with reference to theaccompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As discussed hereinabove, it has been recently reported thatwell-defined, synthetically prepared oxidized phospholipids can regulatethe immune response to oxidized LDL and are thus highly effective intreating atherosclerosis and related diseases, as well as autoimmunediseases and inflammatory disorders. It has been further reported thatgenerally, etherified oxidized phospholipids are superior to comparableesterified oxidized phospholipids as therapeutic agents.

These highly beneficial oxidized phospholipids typically include aglycerolic backbone, to which a lipid residue, a phosphate residue andan oxidized moiety-containing lipid residue are attached, as isdescribed in detail, for example, in U.S. Pat. No. 6,838,452 and in WO04/106486.

As is further discussed hereinabove, the presently known methods ofpreparing such well-defined synthetic oxidized phospholipids involvemulti-step syntheses. While these multi-step syntheses were found to berelatively efficient, resulting in moderate to good yield, these methodsare limited by the need to perform laborious isolation and purificationprocedures of the various intermediates formed throughout the syntheses.Particularly, these procedures typically involve techniques such ascolumn chromatography, which, as is widely recognized by a skilledartisan, is industrially inapplicable, or at least inefficient in termsof costs, complexity and use of excessive amounts of organic solvents,which may be hazardous and requires special care of the waste disposal.The need to use column chromatography in these methods stems from thefact that the intermediates, as well as the final products formed duringthese multi-step syntheses, cannot be isolated and/or purified by moreconventional techniques such as extraction, crystallization and thelike.

Since such synthetically-prepared oxidized phospholipids exhibitexceptionally beneficial therapeutic activity, it is highly desired toprepare these compounds in a high level of purity. Furthermore, sincethe preparation of such oxidized phospholipids involves multi-stepsyntheses, purification of the intermediates is required in order toperform such a process is reasonable yields and with minimal amount ofside products.

In a search for novel methods of preparing oxidized phospholipids, whichcould be efficiently utilized in the scaled-up production of thesecompounds, while circumventing the need to use laborious techniques suchas column chromatography, the present inventors have designed andsuccessfully practiced novel synthetic methodologies for introducing anoxidized moiety and/or a phosphate moiety to compounds that have aglycerolic backbone, which circumvent the disadvantageous use of columnchromatography and which result in relatively high yield of purecompounds. The methods described herein further typically utilizecommercially available, non-hazardous reactants, which further providesfor the industrial applicability thereof.

The novel synthetic methodologies described herein can be divided asfollows:

(i) a novel method of introducing an oxidized moiety to a compoundhaving a glycerolic backbone, via introduction of an unsaturated moietyand oxidation of the unsaturated moiety, whereby upon said oxidation theoxidized moiety-containing compound is isolated and purified by means ofa water-soluble adduct;

(ii) a novel method of introducing an oxidized moiety to a compoundhaving a glycerolic backbone, via introduction of an unsaturated moietyand oxidation of the unsaturated moiety, whereby said oxidation isperformed via an epoxide intermediate and in the presence of a selectiveprotecting group;

(iii) a novel method of introducing an oxidized moiety to a compoundhaving a glycerolic backbone, via introduction of an unsaturated moietyand oxidation of the unsaturated moiety, whereby said oxidation isperformed directly and allows isolation and purification of the oxidizedproduct by simple phase-separation means;

(iii) a novel method of introducing an oxidized moiety to a compoundhaving a glycerolic backbone, via direct introduction of the oxidizedmoiety; and

(iv) a novel method of introducing a phosphate moiety to a glycerolipidoptionally having an oxidized or pre-oxidized moiety attached thereto,via introduction of a reactive phosphorous-containing group.

Due to the superior performance of oxidized phospholipids in which theoxidized moiety-containing residue is attached to the backbone via anether bond, these methods are all directed for the attachment of theoxidized moiety-containing residue to the glycerolic backbone via anether bond.

As is demonstrated in the Examples section that follows, using thesemethodologies, well-defined oxidized phospholipids, have beensuccessfully prepared in relatively high yield and purity.

Thus, according to one aspect of the present invention there is provideda method of preparing a compound having a glycerolic backbone and atleast one oxidized moiety-containing residue attached to the glycerolicbackbone via an ether bond, which is devoid of column chromatography.The method, according to this aspect of the present invention, iseffected by:

providing a first compound having a glycerolic backbone and at least onefree hydroxyl group;

providing a second compound having at least one unsaturated bond and atleast one reactive group capable of forming an ether bond with said freehydroxyl group;

reacting the first compound and the second compound to thereby obtain athird compound, which has a glycerolic backbone and an unsaturatedbond-containing residue being attached to the glycerolic backbone via anether bond;

isolating the third compound, to thereby obtain a purified thirdcompound;

reacting the purified third compound with an oxidizing agent, to therebyobtain a fourth compound, which has a glycerolic backbone and anoxidized moiety-containing residue attached to the glycerolic backbonevia an ether bond; and

isolating the fourth compound to thereby obtain a purified fourthcompound, thereby obtaining the compound having a glycerolic backboneand at least one oxidized moiety-containing residue attached to theglycerolic backbone via an ether bond.

As used herein throughout, the phrase “a compound having a glycerolicbackbone”, which is also referred to herein interchangeably as “aglycerolic compound”, or a “glycerol compound” describes a compound thatincludes the following skeleton:

When the compound is glycerol, each of the glycerolic positions sn-1,sn-2 and sn-3 is substituted by a free hydroxyl group.

As used herein throughout, the phrases “oxidized moiety” and “anoxidized moiety-containing residue”, which are used hereininterchangeably, describe an organic moiety in which at least one of itscarbon atoms is substituted by an oxygen atom. Examples, withoutlimitation, include aldehyde, carboxylic acid, carboxylic ester, diol,acetal, and ketal. The phrases “a compound having an oxidizedmoiety-containing residue” and “an oxidized moiety-containing compound”are also used herein interchangeably.

The method according to this aspect of the present invention is based onintroducing an unsaturated moiety to the glycerolic compound andsubjecting the unsaturated bond to oxidative cleavage. However, whilesuch a synthetic route has been employed in the presently knownsyntheses of glycerolic oxidized phospholipids, the present inventorshave now designed and successfully practiced such a process in which theglycerolic compound that has an oxidized moiety attached thereto can beisolated and purified without using column chromatography.

Introduction of the unsaturated moiety to the glycerolic compound istypically performed using methods known in the art, such as described,for example, in U.S. Pat. No. 6,838,452.

Typically, a first compound, which has a glycerolic backbone and atleast one free hydroxyl group, is selected as the starting material.

A compound that has an unsaturated moiety and a first reactive group,which is also referred to herein as the second compound, is obtained,either commercially or using methods known in the art, and is reactedwith the glycerolic starting material.

The first reactive group is selected capable of reacting with the freehydroxyl group. Reacting with the free hydroxyl group so as to form anether bond is typically performed via a nucleophilic mechanism andtherefore the first reactive group is preferably characterized as a goodleaving group and can be, for example, halide, sulfonate, and any otherleaving group.

Preferably, the reactive group is halide and more preferably, it isbromide.

The second compound is preferably selected such that the unsaturatedmoiety is present at a terminus position thereof, so as to facilitatethe oxidation reaction that follows. By “unsaturated moiety” it is meantherein a moiety that includes at least two carbon atoms that are linkedtherebetween by an unsaturated bond, e.g., a double bond or a triplebond, preferably a double bond.

Further preferably, the second compound comprises from 4 to 30 carbonatoms, more preferably from 4 to 27 carbon atoms, more preferably from 4to 16 carbon atoms, more preferably from 4 to 10 carbon atoms, morepreferably from 4 to 8 carbon atoms, and most preferably the secondcompound comprises 6 carbon atoms.

Reacting the first compound and the second compound described herein istypically performed in the presence of a base. Suitable bases for use inthis context of the present invention include, without limitation,inorganic bases such as sodium hydroxide, lithium hydroxide, calciumhydroxide, barium hydroxide and potassium hydroxide.

Reacting the first compound and the second compound is typicallyperformed in the presence of a solvent. Suitable solvents for use inthis context of the present invention include, without limitation, nonpolar solvents such as petrol ether, hexane, benzene and toluene.

In cases where it is desired to perform the reaction selectively,namely, introducing the unsaturated moiety to a certain position of theglycerolic backbone, free hydroxyl group other than the reactinghydroxyl, if present, should be protected prior to the reaction.

Thus, in such cases, the method according to this aspect of the presentinvention optionally and preferably further comprises, prior to reactingthe first compound and the second compound, protecting one or moreadditional free hydroxyl groups that may be present within the firstcompound.

Any of the known hydroxyl-protecting groups can be used in this contextof the present invention. According to preferred embodiment of thisaspect of the present invention, the protecting group is trityl.

Trityl is a bulky group, which typically serves as a selectiveprotecting group, due to steric hindrance. Thus, while reacting aglycerolic compound that has more than one free hydroxyl group,typically, the trityl group would be reacted with the less hinderedgroup.

As noted hereinabove and is further discussed in detail in U.S. Pat. No.6,838,452 and in WO 04/106486, the position of the glycerolic backboneto which an oxidized moiety is attached affects the activity of thecompound. It is therefore highly beneficial to perform the preparationof the glycerolic compounds described herein selectively, such that theoxidized moiety-containing residue would be attached to the desiredposition. As is further demonstrated in U.S. Pat. No. 6,838,452,oxidized phospholipids that have an oxidized moiety-containing residueattached to the sn-2 position of the glycerol backbone exhibit asuperior performance.

To that end, the use of trityl group as the protecting group whileintroducing the above-described second compound to the glycerolicbackbone is highly beneficial, since due to its bulkiness, protection ofthe hydroxyl end groups, at the sn-1 and/or an-3 positions would beeffected, leaving the hydroxyl group at the sn-2 available for furthersubstitutions.

Once the reaction between the first compound and the second compound iscompleted, a reaction mixture which contains a compound that has aglycerolic backbone and an unsaturated moiety-containing residueattached thereto via an ether bond is obtained. Such a compound is alsoreferred to herein interchangeably as a third compound.

Depending on the starting material used, the third compound can furtherinclude one or more protecting groups, protecting free hydroxyl groupsthat may be present within the glycerolic backbone.

The third compound, either protected or deprotected, is then isolatedfrom the reaction mixture and treated so as to obtain a purifiedcompound.

In a preferred embodiment, isolating the third compound is performed byfirst collecting the formed third compound. Collecting the thirdcompound is typically performed using conventional techniques such asextraction, removal of the solvent, filtration and the like, includingany combination thereof. Once collected, the crude product is dissolvedis a solvent, whereby the solvent is selected such that the thirdcompound is soluble therein whereby impurities formed during thereaction between the first and the second compounds are insolubletherein.

The term “impurities” is used herein to describe any substance that ispresent in the final crude product and is not the product itself andinclude, for example, unreacted starting materials and side products.

Using such a solvent, a mixture that includes a solution of the thirdcompound in such a solvent and insoluble substances is obtained.Suitable solvents for use in this context of the present inventioninclude, without limitation, non-polar solvents such as petrol ether,hexane, benzene, heptane and toluene. Preferably, the solvent is petrolether. Further preferably, the solvent is hexane.

The insoluble impurities are then removed from the mixture, preferablyby filtration, the solvent is removed and a purified third compound isobtained while circumventing the need to use column chromatography inthe purification procedure thereof.

The purified third compound is then reacted with an oxidizing agent, soas to oxidize the unsaturated moiety and thereby obtain a fourthcompound, in which an oxidized moiety-containing residue is attached tothe glycerolic backbone via an ether bond.

The oxidizing agent is selected depending on the desired oxidizedmoiety, as is detailed hereinbelow, and can be, for example, a peroxide,a periodate, a bismuthate, a permanganate, a chlorite, ozone, silveroxide, osmium tetraoxide and any combination thereof.

As used herein, the term “periodate” describes a compound having theformula XIO₄, wherein X can be hydrogen (for periodic acid) or amonovalent cation of a metal (e.g., sodium, potassium). A preferredperiodate is sodium periodate (NaIO₄).

The term “bismuthate” describes a compound having the formula XBiO₃,wherein X can be hydrogen or a monovalent cation of a metal (e.g.,sodium, potassium).

The term “permanganate” describes a compound having the formula XMnO₄,wherein X can be hydrogen or a monovalent cation of a metal (e.g.,sodium, potassium). Preferred permanganate is potassium permanganate(KMnO₄).

The term “chlorite” describes a compound having the formula XClO₂,wherein X can be hydrogen or a monovalent cation of a metal (e.g.,sodium, potassium). As used herein, the term “peroxide” include acompound having the formula R—O—O—H, wherein R can be hydrogen, alkyl,cycloalkyl, aryl, oxyalkyl, oxycycloalkyl and oxyaryl, as these termsare defined herein.

As used herein throughout, the term “alkyl” refers to a saturatedaliphatic hydrocarbon including straight chain and branched chaingroups. Preferably, the alkyl group has 1 to 20 carbon atoms.

A “cycloalkyl” group refers to an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl.

The terms “oxyalkyl”, “oxycycloalkyl” and “oxyaryl” describe anR′—C(═O)— group, whereby R′ is alkyl, cycloalkyl or aryl, respectively,such that the peroxide is a peroxycarboxylic acid.

Preferably, the peroxide is hydrogen peroxide or a peroxycarboxylic acid(e.g., perbenzoic acid).

Thus, in one embodiment of this aspect of the present invention, theoxidized moiety is aldehyde and reacting the third compound with anoxidizing agent is performed by first converting the unsaturated moietyin the third compound to a diol moiety, preferably by means of anoxidizing agent, which is referred to herein as a first oxidizing agent;and then further oxidizing the diol moiety, by means of a secondoxidizing agent, to the aldehyde moiety.

The first and the second oxidizing agents can be the same or differentand can be, for example, a peroxide, a periodate, a bismuthate, apermanganate, a chlorite, ozone and any combination thereof.

In cases where the first and the second oxidizing agents are the same,and depending on the oxidizing agent used, converting the unsaturatedmoiety to a diol moiety and oxidizing the diol moiety can be performedsimultaneously. Suitable oxidizing agents that can be used in thisrespect include oxidizing agents that are capable of inducing anoxidative cleavage of an unsaturated moiety such as, for example, ozone,osmium tetraoxide, and potassium permanganate.

In cases where the first and the second oxidizing agents are different,preferably the first oxidizing agent is a peroxide, such as hydrogenperoxide and the second oxidizing agent is, for example, a periodate ora bismuthate.

The reaction conditions at which the converting and oxidizing proceduresare performed are determined in accordance with the oxidizing agentused.

In a preferred embodiment of this aspect of the present invention, thefirst and the second oxidizing agents are different and converting theunsaturated moiety to a diol moiety and oxidizing the diol moiety areperformed sequentially. Further according to a preferred embodiment ofthis aspect of the present invention, once the diol is obtained, theprotecting group, if present, is removed so as to obtained a compoundhaving three or more free hydroxyl groups (herein, a triol). Such acompound can be easily purified, prior to its oxidation to an aldehyde,by means of crystallization, due to its unique chemical features, as isdemonstrated in the Examples section the follows (see, Example 1). Oncepurified, selective protection the free hydroxyl group at the sn-1and/or sn-3 positions can be effected, prior to the next synthetic step.

The thus formed aldehyde-containing glycerolic compound, is thenisolated from the reaction mixture and purified.

In a preferred embodiment of this aspect of the present invention, thealdehyde is purified by means of forming a water-soluble adduct thereof.

Thus, once the reaction with the oxidizing agent(s) is completed, thealdehyde-containing fourth compound is collected using conventionaltechniques as described herein above and thereafter the crude product isconverted into a water-soluble adduct thereof. By performing such aconversion in a biphasic system, an aqueous phase that contains thewater-soluble adduct and an organic phase, which containswater-insoluble impurities are obtained. Since most of the side productsand unreacted material formed during the oxidation reaction are organicsubstances, such substances are easily separated from the water-solubleadduct by collecting the aqueous phase. The aldehyde-containing compoundis thereafter recovered by decomposing the water-soluble adduct.

Suitable water-soluble adducts that can be used in this context of thepresent invention are preferably obtained by reacting thealdehyde-containing compound with a Girard reagent.

Girard reagents are a family of substances that are capable of formingwater-soluble hydrazone adducts with carbonyl-containing compounds, andthus allow the separation of carbonyl-containing compounds from otherorganic non-carbonylic compounds. Girard reagents are ionic derivativesof semicarbazide.

The T form is (Carboxymethyl)trimethylammonium chloride hydrazide:

The D form is (Carboxymethyl)dimethylammonium chloride hydrazide:

And the P form is 1-(Carboxylmethyl)pyridinium chloride hydrazide:

Thus, by converting the aldehyde-containing compound to a water-solubleadduct thereof with a Girard reagent, a purified fourth compound iseasily and conveniently obtained, while avoiding the use of columnchromatography.

In cases where the oxidized moiety is a carboxylic acid, reacting thethird compound with an oxidizing agent can be performed by firstproviding an aldehyde-containing compound, optionally and preferably asdescribed hereinabove, and further optionally and preferably, byproviding a purified aldehyde-containing compound, using the methodologydescribed hereinabove, and thereafter further oxidizing the aldehyde tocarboxylic acid.

Oxidizing the aldehyde to a carboxylic acid is preferably performed byreacting the aldehyde with an oxidizing agent such as chlorite.

Alternatively, the unsaturated moiety can be oxidized to a carboxylicacid via an epoxide intermediate.

Thus, reacting the third compound with an oxidizing agent can beperformed by converting the unsaturated moiety to epoxide, andconverting the epoxide to the carboxylic acid. Preferably, convertingthe epoxide to a carboxylic acid is performed by converting the epoxideto diol and oxidizing the diol so as to obtain the carboxylic acidmoiety.

Converting the third compound to an epoxide is preferably performed byreacting the third compound with a peroxide, as defined hereinabove, andmore preferably with a peroxycarboxylic acid.

Converting the epoxide to diol is preferably performed by reacting theepoxide with perchloric acid (HClO₄). Alternatively, the epoxide isconverted to diol by reacting it with sulfuric acid.

The diol is then converted to the carboxylic acid by reacting it with athird oxidizing agent. The third oxidizing agents can be selected fromperiodate, bithmutate, permanganate, chlorite and any combinationthereof. Preferably, the diol is converted to the carboxylic acid byreacting it with a periodate, followed by a chlorite.

The fourth compound thus obtained, having a glycerolic backbone and acarboxylic acid-containing moiety attached thereto via an ether bond, inthen purified, so as to obtain a purified product.

The present inventors have now surprisingly found that a fourth compoundobtained via the epoxide intermediate can be easily purified, whileavoiding the use of column chromatography, if a free hydroxyl groupthereof is protected by a protecting group such as acetate, pivaloate orbenzoate.

As mentioned hereinabove, a free hydroxyl group, if present in theglycerol backbone, is preferably protected, whereby a preferable,selective protecting group is trityl. However, since trityl is a large,bulky and non-polar moiety, its presence might, in some cases,complicate the isolation and purification procedures of the variousintermediates and the final product.

The present inventors have now uncovered that limitations associatedwith the trityl group can be readily circumvented by: (i) isolating analdehyde-containing compound via the formation of a water-soluble adductthereof, as is widely described hereinabove; or (ii) replacing thetrityl protecting group by a less bulky group, subsequent to theintroduction of the second compound. In addition, as describedhereinabove, when oxidizing the third compound comprises the formationof a diol, once the diol is forms, the trityl protecting group can beremoved and the resulting triol can be isolated by means ofcrystallization.

Thus, according to a preferred embodiment of the present invention, theprocess further comprises, subsequent to the provision of the purifiedthird compound and/or prior to reacting the third compound with anoxidizing agent: replacing the trityl group with a protecting groupselected from the group consisting of acetate, pivaloate or benzoate.

Replacing the trityl protecting group is typically effected by removingthe trityl group, so as to obtain a free hydroxyl group and protectingthe hydroxyl group with the desired protecting group.

Protecting the hydroxyl group with an acetate group is readily performedby reacting the third compound with e.g., acetic anhydride. Protectingthe hydroxyl group with a pivaloate group is readily performed byreacting the third compound with e.g., pivaloyl chloride. Protecting thehydroxyl group with a benzoate group is readily performed by reactingthe third compound with e.g., benzoyl chloride.

Purifying a fourth compound, as described herein, which has an acetate,pivaloate or benzoate protecting group can be carried out byconventional extraction techniques, preferably while using silica gelduring the extraction procedure.

As is demonstrated in the Examples section that follows (see, Example2), it was found that preparing a glycerolic compound having an oxidizedmoiety-containing group attached thereto via an ether bond, via theformation of an epoxide-containing intermediate that has an acetateprotecting group, resulted in highly purified compound and high reactionyield.

The present inventors have further uncovered that a fourth compoundhaving a carboxylic acid as an oxidized moiety can be readily obtainedby reacting the third compounds described herein with a mixture of aperiodate and a permanganate as an oxidizing agent.

Converting the third compound directly to a carboxylic acid-containingcompound is highly beneficial since it evidently render the entireprocess more efficient by reducing the number of synthetic steps andfurther circumvents the need to purify the intermediates formed duringthe oxidation process. In addition, the oxidizing agent utilized in thisroute comprises safe, non-hazardous agents.

Hence, according to the presently most preferred embodiment of thepresent invention, the oxidized moiety is carboxylic acid and oxidizingthe third compound is effected by reacting the third compound with amixture of a periodate and a permanganate.

Such a reaction is preferably performed in the presence of a base.Preferred bases that are suitable for use in this embodiment of thepresent invention include sodium carbonate and sodium bicarbonate.

In cases where the obtained fourth compound has a protecting group, asdescribed hereinabove, once the fourth compound is obtained, isolatedand optionally purified, the protecting group is removed.

In cases where the oxidized moiety is a carboxylic acid, the fourthcompound can be readily isolated upon removal of the protecting groupand obtaining a compound that has a carboxylic moiety and a hydroxymoiety.

Similarly to the procedure described hereinabove for isolating andpurifying the third compound, the fourth compound can be readilypurified by dissolving it in a solvent, whereby the solvent is selectedsuch that the fourth compound is soluble therein whereby impuritiesformed during the oxidation process are insoluble therein.

Moreover, such a solvent can be selected such that the fourth compoundis soluble therein whereby the protecting group is insoluble therein.Thus, performing the removal of the protecting group under conditionsthat involve such a solvent allows removing both the protecting groupand the impurities formed during the oxidation reaction within the samesynthetic step.

Using such a solvent, a mixture that includes a solution of the fourthcompound in such a solvent and insoluble substances such as impuritiesand the protecting group is obtained. Suitable solvents for use in thiscontext of the present invention include, without limitation, non-polarsolvents such as petrol ether, hexane, benzene, heptane and toluene,semi-polar solvents such as ethyl acetate and mixtures thereof.Preferably, the solvent is petrol ether or hexane and/or a mixture ofthereof with ethyl acetate.

The insoluble impurities are then removed from the mixture, preferablyby filtration, the solvent is removed and a purified fourth compound isobtained while circumventing the need to use column chromatography inthe purification procedure thereof and further circumventing the needfor multiple purification procedures of the various intermediatesformed.

In cases where the oxidized moiety is an ester, the process is effectedby providing a carboxylic-acid containing compound and then convertingthe carboxylic acid to the ester. This can be readily carried out, usingprocedures well known in the art. Exemplary procedures are described inthe Examples section that follows (see, for example, Examples 1, 6 and7).

As is discussed hereinabove, compounds having a glycerolic backbone andat least one oxidized moiety-containing residue attached to theglycerolic backbone and further having a phosphorous-containing moietyattached to the glycerolic backbone, preferably a phosphate-containingmoiety, are known as oxidized phospholipids and are highly beneficial intreating various conditions. Thus, the process described hereinoptionally and preferably further comprises introduction of such aphosphorous-containing moiety to the glycerolic backbone.

As used herein, the phrase “phosphorous-containing moiety” describes amoiety, as defined herein, which includes one or more phosphor atoms.Representative examples include, without limitation, phosphates,phosphonates, phosphines, phosphine oxides, phosphites, pyrophosphatesand like.

As used herein the term “phosphonate” describes a —P(═O)(OR′)(OR″)group, where R′ and R″ are each independently hydrogen, or substitutedor unsubstituted alkyl, cycloalkyl or aryl, as defined herein.

The term “phosphinyl” describes a —PR′R″ group, with R′ and R″ asdefined hereinabove.

The term “phosphine oxide” describes a —P(═O)(R′)(R″) end group or a—P(═O)(R′)— linking group, as these phrases are defined hereinabove,with R′ and R″ as defined herein.

The term “pyrophosphate” describes an—O—P(═O)(OR′)—O—P(═O)(OR′)(OR″)(OR′″) group, with R′, R″ as definedherein, and R′″ is defined as R′ or R″.

The term “phosphite” describes an —O—PH(═O)(OR′) group, with R′ asdefined herein.

The term “phosphate” describes an —O—P(═O)₂(OR′) group, with R′ asdefined herein.

The term “thiophosphate” describes an —O—P(═O)(═S)(OR′) group, with R′as defined herein.

The introduction of a phosphorous-containing moiety to the glyceroliccompound can be performed either prior to reacting the first compoundand the second compound, prior to isolating the third compound, prior toreacting the third compound with the oxidizing agent, prior to isolatingthe fourth compound or subsequent to isolating the fourth compound, andcan be performed using any of the methods known in the art.

Introduction of a phosphorous-containing moiety to a compound having aglycerolic compound is therefore performed by:

reacting any of the first compound, the third compound, the purifiedthird compound, the fourth compound or the purified fourth compounddescribed above, with a phosphorous-containing moiety, so as to obtain acompound having a glycerolic backbone and at least one oxidizedmoiety-containing residue attached to the glycerolic backbone andfurther having a phosphorous-containing moiety attached to theglycerolic backbone.

According to a preferred embodiment of the present invention, thephosphorous-containing moiety is a phosphate moiety which is attached tothe glycerolic backbone via a phosphodiester bond.

Thus, the phosphorous-containing moiety can be, for example, phosphoricacid, phosphoryl choline, phosphoryl ethanolamine, phosphoryl serine,phosphoryl cardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine and phosphoglycerol.

Preferably, the phosphorous-containing moiety is attached to the sn-3position of the glycerolic backbone and thus, introduction of such amoiety is performed selectively, by appropriately protecting other freehydroxyl groups that are present in the reacting compound ordeprotecting a protected hydroxyl group at the desired position.

In the presently known methods of preparing oxidized phospholipids, thephosphorous-containing moiety is typically introduced prior to theprovision of an oxidized-moiety containing compound.

In addition, in cases where the phosphorous-containing moiety isphosphoryl choline, a widely used and beneficial moiety in suchcompounds, the presently known methods involve N-alkylation reactions,which involve hazardous and environmentally unfriendly reagents such as,for example, trimethylamine.

The present inventors have now uncovered that (i) aphosphorous-containing moiety can be readily introduced subsequent tothe provision of an oxidized moiety-containing compound; and (ii) theintroduction of the phosphorous-containing moiety can be efficientlyperformed via a reactive phosphorous-containing intermediate.

Based on the above, the present inventors have designed and successfullypracticed a novel process for introducing a phosphorous-containingmoiety to compounds having a glycerolic backbone and an oxidizedmoiety-containing residue attached thereto via an ether bond.

This process, combined with the process described above for preparingthe oxidized moiety-containing compound, can be beneficially used forpreparing the therapeutically beneficial oxidizes phospholipidsdescribed above.

Thus, according preferred embodiments of the present invention, theintroduction of the phosphorous-containing moiety is performedsubsequent to the production of the third compound or subsequent to theproduction of the fourth compound, with the latter being preferred.However, it should be noted that the process of introducing thephosphorous-containing moiety presented herein is also applicable at anyother stage.

The introduction of a phosphorous-containing moiety to a glyceroliccompound is therefore preferably effected, according to the presentembodiments, by reacting a first compound, a third compound, a purifiedthird compound, a fourth compound or a purified fourth compound asdescribed above, which has a free hydroxyl group, with a reactivephosphorous-containing compound, so as to produce a compound having areactive phosphorous-containing group; and converting the reactivephosphorous-containing group to the phosphorous-containing moiety.

The reactive phosphorous-containing compound is selected such that uponsaid reacting, a reactive phosphorous-containing group attached to theglycerolic backbone is obtained. The reactive phosphorous-containingcompound is therefore selected as having a second reactive group and athird reactive group, whereby the second reactive group is selectedcapable of reacting with the free hydroxyl group and the third reactivegroup is selected capable of being converted to thephosphorous-containing moiety.

Reactive groups that are capable of reacting with a free hydroxyl groupsinclude, for example halides, sulfonyl chlorides, acyl halides and thelike.

Preferably the second reactive group is halide and more preferably it ischloride.

While as described hereinabove, preferable phosphorous-containingmoieties are phosphate moieties, converting the phosphorous-containingcompound to the desired phosphorous-containing moiety typically involvesa formation of a phosphate-ester bond. Such a bond can be obtained, forexample, by reacting a phosphoric derivative such as phosphoryl chloridewith a hydroxy-containing moiety.

Thus, according to a preferred embodiment, the reactivephosphorous-containing compound is phosphorous oxychloride (POCl₃), suchthat the third and the second reactive groups are both chlorides and thecompound having a phosphorous-containing reactive group has a glycerolicbackbone and a phosphoryl chloride residue attached thereto.

Reacting the first compound, the third compound, the purified thirdcompound, the fourth compound or the purified fourth compound with thephosphorous oxychloride is typically carried out in the presence of abase. Suitable bases include organic and inorganic bases, with organicbases being preferred. Thus, the reaction is preferably effected inpresence of a base such as, for example, trialkylamine (e.g.,triethylamine).

This reaction is further preferably carried out in the presence of asolvent, preferably a polar solvent such as THF.

The phosphoryl chloride-containing glycerolic containing compoundobtained by the process described herein can be readily converted to anydesired phosphorus-containing moiety and is therefore a highlybeneficial intermediate.

Thus, for example, it can be converted to phosphoric acid by a simplehydrolysis thereof, as is exemplified in the Examples section thatfollows.

Alternatively, it can be reacted with a hydroxy-containing moiety, andoptionally and preferably also with water, to thereby obtain otherphosphate moieties.

Preferred phosphate moieties that are incorporated in therapeuticoxidized phospholipids (e.g., phosphoryl choline, phosphorylethanolamine) typically include an aminoalkyl group, which can befurther N-alkylated.

Converting the phosphoryl chloride intermediate to such phosphatemoieties can thus be readily performed by reaction with a derivative ofthe desired aminoalkyl group, selected capable of reacting with thethird reactive group (being a chloride).

Thus, for example, aminoalkyl-containing phosphate moieties can beobtained by reacting the phosphoryl chloride intermediate with anaminoalcohol. If desired, the aminoalcohol can thereafter be furtheralkylated, so as to produce an N-alkylated aminoalkyl phosphate moiety,as in the case of a phosphoryl choline moiety.

Obtaining such an N-alkylated aminoalkyl phosphate moiety attached to aglycerolic backbone using the process described above is highlybeneficial since it circumvents the need to use hazardous materials suchas the trimethylamine typically used for obtaining such compounds.

As discussed hereinabove, the introduction of the phosphorous-containingmoiety can be performed either prior to or subsequent to theintroduction of an oxidized moiety-containing residue to the glyceroliccompound. As is demonstrated in the Examples section that follows, aphosphoryl choline moiety was successfully introduced into glyceroliccompounds having either an oxidized-moiety containing residue or anunsaturated-moiety containing residue (see, Examples 4 and 5). Thus, theprocess of introducing a phosphate moiety via a reactivephosphorous-containing intermediate presented herein can be performedeither with glycerolic compounds having an oxidized or pre-oxidizedmoiety attached thereto via an ether bond.

In their search for improved methods for preparing oxidizedphospholipids, the present inventors have further designed and practicedan additional process for preparing a glycerolic compound having anoxidized moiety attached thereto via an ether bond, which is effected bydirect introduction of an oxidized moiety-containing residue to aglycerolic compound.

Hence, according to another aspect of the present invention, there isprovided a method of preparing a compound having a glycerolic backboneand at least one oxidized moiety-containing residue attached to theglycerolic backbone via an ether bond, which is effected by:

providing a first compound having a glycerolic backbone and at least onefree hydroxyl group, as described hereinabove;

providing a fifth compound having at least one oxidized moiety, asdescribed hereinabove, and at least one fourth reactive group;

reacting the first compound and the fifth compound to thereby obtain areaction mixture containing a sixth compound, being the compound havinga glycerolic backbone and at least one oxidized moiety-containingresidue attached to the glycerolic backbone via an ether bond; and

isolating the compound having a glycerolic backbone and at least oneoxidized moiety-containing residue attached to the glycerolic backbonevia an ether bond.

The process according to this aspect of the present invention thereforeinvolves the reaction of the first compound described hereinabove with acompound that has a reactive group that is capable of reacting with afree hydroxyl group of the first compound, referred to herein as afourth reactive group, and an oxidized moiety. Such a compound isreferred to herein as the fifth compound.

The oxidized moiety in the fifth compound can be any of the oxidizedmoieties described above, namely, an aldehyde, a diol, a carboxylicacid, an ester an acetal or a ketal. Optionally, the oxidized moiety canbe a semi-oxidized moiety, namely, being readily converted to a desiredoxidized moiety without reacting with an oxidizing agent. An example ofsuch a semi-oxidized moiety is nitrile, which can be readily convertedto a carboxylic acid by a simple hydrolysis.

The fourth reactive group in the fifth compound is as described hereinfor the first reactive group and is preferably a halide and morepreferably a bromide.

Reacting the first compound and the fifth compound is preferablyeffected in the presence of a base. Relatively strong inorganic basessuch as, for example, sodium hydride, potassium hydroxide, lithiumaluminum hydride, sodium amide, sodium hydroxide and any mixture thereofare preferred.

Under such reaction conditions, a fifth compound which has 4 or 5 carbonatoms might be cyclized during the reaction, thus adversely affectingthe reaction efficiency.

Thus, preferably, the fifth compound preferably has less than 4 or morethan 5 carbon atoms.

As described hereinabove, in cases where the first compound has morethan one hydroxyl group attached thereto, the hydroxyl group isoptionally and preferably protected by a protecting group, prior toreacting the first and the fifth compounds.

Once the sixth compound is obtained, the protecting group can be removedand the compound is purified using conventional purification methods.

The process according to this aspect of the present invention is highlybeneficial since it enables to prepare the described oxidizedmoiety-containing compound in a one-step synthesis.

Using this process, oxidized phospholipids can be readily obtained byintroducing a phosphorous-containing moiety, a described in detailhereinabove, either prior to or subsequent to the reaction with thefifth compound. The introduction of the phosphorous-containing moiety ispreferably performed using the process presented hereinabove.

In any of the processes described herein, the first compound can includean alkylene chain attached thereto. Preferably, the alkylene chain isattached to the sn-1 position of the first compound.

The alkylene chain can be attached to the glycerolic compound by, forexample, an ester bond or an ether bond. Preferably, the alkylene chainis attached via an ether bond, such that the final product is adietherified glycerolic compound.

Thus, in each of the processes described herein, the first compound is aglycerolipid, as defined herein and preferably, a mono-etherifiedglycerolipid in which the lipid moiety is attached to the sn-1 positionof the glycerol. Such a first compound therefore has one free hydroxylgroup, which, as described hereinabove, is preferably protected prior toany reaction.

Thus, the first compound can be, for example, a glycerol, aglycerolipid, a mono-etherified glycerolipid, a di-etherifiedglycerolipid, a phosphoglycerol, a phosphoglyceride, a mono-etherifiedphosphoglyceride and a lysolechitin.

As is discussed in detail hereinabove, the position at which theoxidized moiety-containing residue is attached to the glycerolicbackbone affects the activity of the resulting compounds and thus, as isfurther discussed hereinabove, it is preferably to perform the reactionselectively.

Preferably, in any of the processes described herein the oxidizedmoiety-containing residue is attached to the sn-2 position of thecompound. Thus, by appropriately selecting and/or protecting the firstcompound, selective attachment of the oxidized moiety-containing residueis performed.

In a preferred embodiment of the present invention, the first compoundtherefore has the following general formula I:

wherein:

A₁ is absent or is selected from the group consisting of CH₂, CH═CH andC═O;

R₁ is selected from the group consisting of H and a hydrocarbon chainhaving from 1 to 30 carbon atoms; and

R₃ is selected from the group consisting of hydrogen, alkyl, aryl,phosphoric acid, phosphoryl choline, phosphoryl ethanolamine, phosphorylserine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylserine, phosphatidyl cardiolipin, phosphatidyl inositol, phosphorylcardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, phosphoglycerol.

Using any of the processes described hereinabove, a compound having thefollowing general Formula II can thus be obtained:

wherein:

A₁ is selected from the group consisting of CH₂, CH═CH and C═O and ispreferably CH₂;

A₂ is CH₂;

R₁ is an alkyl having 1-30 carbon atoms;

R₂ is

whereas:

X is an alkyl chain having 1-24 carbon atoms;

Y is selected from the group consisting of hydrogen, hydroxy, alkyl,alkoxy, halide, acetoxy and an aromatic functional group; and

Z is selected from the group consisting of:

with R₄ being an alkyl or aryl; and

R₃ is selected from the group consisting of hydrogen, alkyl, aryl,phosphoric acid, phosphoryl choline, phosphoryl ethanolamine, phosphorylserine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylserine, phosphatidyl cardiolipin, phosphatidyl inositol, phosphorylcardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, phosphoglycerol.

As is demonstrated in the Examples section that follows, theabove-described processes can be used for producing oxidizedphospholipids, and particularly therapeutically beneficial oxidizedphospholipids such as 1-hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine(also known in the art and referred to herein as CI-201). For example,using the process described in Example 6 hereinbelow,1-Hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine was produced in anindustrial scale of dozens of Kg.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

General Synthetic Pathways:

According to the teachings of the present invention, several generalsynthetic concepts are used for preparing oxidized phospholipids, asfollows:

(i) Preparation of a glycerolipid compound having at least one oxidizedmoiety-containing residue attached thereto via an ether bond, byattachment of an unsaturated residue to a glycerolipid and oxidizing theunsaturated bond, while using a Girard reagent and/or crystallization ofa triol-containing compound for isolating the oxidized product, asexemplified in Example 1 and Schemes I-V;

(ii) Preparation of a glycerolipid compound having at least one oxidizedmoiety-containing residue attached thereto via an ether bond, byattachment of an unsaturated residue to a glycerolipid and oxidizing theunsaturated bond via an epoxide intermediate, while using an acetoxyprotecting group, as exemplified in Example 2 and Schemes VI-X;

(iii) Preparation of a glycerolipid compound having at least oneoxidized moiety-containing residue attached thereto via an ether bond bydirect introduction of an oxidized moiety-containing compound, asexemplified in Example 3 and Scheme XI; and

(iv) Introduction of a reactive phosphorous-containing moiety to aglycerolipid compound having one or two oxidized (or pre-oxidized)moiety-containing residues attached thereto via an ether bond using areactive phosphorous-containing compound (for example, phosphorousdichloride) for forming a reactive intermediate, as exemplified inExamples 4 and 5 and Schemes XII-XIV.

Example 1 Preparation of rac-1-hexadecyl-2-(5′-pentanoic methylester)-glycerol using periodate and a Girard T reagent

In this example, an unsaturated moiety is introduced into a glycerolicbackbone and is thereafter oxidized by means of formic acid, hydrogenperoxide and periodate. Then thus formed oxidized product is purified bymeans of a Girard reagent.

As a representative example, the preparation ofrac-1-hexadecyl-2-(5′-pentanoic methyl ester)-glycerol is herebydescribed.

rac-1-Hexadecyl-2-(5′-pentanoic methyl ester)-glycerol is prepared inaccordance with the teachings of the present invention, as is describedin Schemes I through V below.

1-Hexadecyl-3-tritylglycerol was prepared as described in U.S. Pat. No.6,838,452. In brief, D-acetone glycerol (4 grams), powdered potassiumhydroxide (approximately 10 grams) and hexadecyl bromide (9.3 grams) inbenzene (100 ml) were stirred and refluxed for 5 hours, while removingthe water formed by azeotropic distillation (compare W. J. Baumann andH. K. Mangold, J. Org. Chem. 29: 3055, 1964 and F. Paltauf, Monatsh.99:1277, 1968). The volume of the solvent was gradually reduced to about20 ml, and the resulting mixture was cooled to room temperature anddissolved in ether (100 ml). The resulting solution was washed withwater (2×50 ml), and the solvent was removed under reduced pressure. A100 ml mixture of 90:10:5 methanol:water:concentrated hydrochloric acidwas added to the residue and the mixture was refluxed for 10 minutes.The product was extracted with ether (200 ml) and was washedconsecutively with water (50 ml), 10% sodium hydroxide (20 ml) and againwith water (volumes of 20 ml) until neutral. The solvent was removedunder reduced pressure and the product (8.8 grams) was crystallized fromhexane to give 7.4 grams of pure 1-hexadecyl-glycerol.

1-Hexadecyloxy-glycerol (7.9 grams), triphenylchloromethane (8.4 grams)and dry pyridine (40 ml) were heated at 100° C. for 12 hours. Aftercooling, 300 ml of ether and 150 ml of ice-cold water were added, andthe reaction mixture was transferred to a separatory funnel. The organicphase was washed consecutively with 50 ml of ice water, 1% potassiumcarbonate solution (until basic) and 50 ml of water, then dried overanhydrous sodium sulfate. The solvent was evaporated, the residue wasdissolved in 150 ml of warm petroleum ether and the resulting solutionwas cooled at 4° C. overnight. After filtration of the precipitate, thefiltrate was evaporated and the residue was recrystallized from 20 ml ofethyl acetate at −30° C., yielding 8.2 grams of1-Hexadecyl-3-tritylglycerol, melting point 49° C.

As depicted in Scheme 1,1-hexadecyl-3-tritylglycerol (14.78 grams,0.0265 mole), 6-bromo-1-hexene (4.85 grams) and powdered potassiumhydroxide (approximately 10 grams) in hexane (200 ml) were stirred andrefluxed for 6 hours, while removing the water formed by azeotropicdistillation. The reaction mixture was cooled to room temperature,washed with water (3×100 ml), and the solvent removed under reducedpressure. The residue was dissolved in chloroform (50 ml) and purifiedby filtration over silica gel 60 (12.5 grams). The chloroform wasremoved under reduced pressure and the residue dissolved in petroleumether (100 ml). The solution was kept at 4° C. for overnight, duringwhich precipitation of byproducts occurred. Filtration and removal ofthe solvent under reduced pressure gave 12.15 grams (0.0190 mole) of1-Hexadecyl-2-(5′-hexenyl)-3-tritylglycerol (72% yield).

1-Hexadecyl-2-(5′-hexenyl)-3-tritylglycerol (19.80 g) was dissolved informic acid (100 ml). The yellow solution was stirred at roomtemperature for 2 hours and was then cooled in ice bath. Hydrogenperoxide 33% (25 ml) was added dropwise to ice-cooled solution during 50minutes. The color of the reaction mixture almost immediately changedfrom yellow to white. After the addition was completed stirring inice-bath was continued for additional 4 hours. The reaction mixture wasthereafter poured on ice (150 grams) and extracted with ether (3×100ml). The orange etheral solution was washed with water (100 ml) and thesolvent was removed under reduced pressure. The residue was dissolved indichloromethane (150 ml), washed with saturated aqueous solution ofsodium bicarbonate (100 ml) and the solvent was removed under reducedpressure. The residue was then dissolved in hot hexane (250 ml).Precipitation of white compound was obtained immediately. The solutionwas maintained at 4° C. overnight. Filtration of the precipitate (0.53grams), followed by removal of the solvent under reduced pressure gave20.03 grams of yellow oily residue. This residue was dissolved iniso-propanol (200 ml) and aqueous solution of sodium hydroxide (17 gramsin 50 ml of water) was added. The resulting solution was heated to 90°C. for 2 hours and was then cooled and poured on ice (150 grams). Thenthe mixture was extracted with dichloromethane (3×100 ml), the organicphase was washed with water (100 ml) and saturated aqueous solution ofsodium dihydrogen phosphate and was dried over anhydrous Na₂SO₄. Afterremoval of the solvent under reduced pressure 10.77 grams of crudeproduct were obtained. The crude product was then dissolved in 80%methanol (100 ml) and the solution was kept at 4° C. overnight.Filtration of the precipitate and removal of most of the solvent underreduced pressure. Extraction with dichloromethane (3×100 ml), dryingover anhydrous Na₂SO₄ and removing of the solvent under reduced pressureRecrystallization from hexane (250 ml) gave 7.44 grams of pure1-Hexadecyl-2-(5′,6′ dihydroxy-hexanyl)-glycerol.

As depicted in Scheme III,1-Hexadecyl-2-(5′,6′-dihydroxy-hexanyl)-glycerol (7.84 grams) wasdissolved in isopropanol (50 ml) and water (12 ml). NaIO₄ (9 grams) wasadded and the reaction mixture was stirred at room temperature for 3hours. Water (50 ml) was added and the reaction mixture extracted withchloroform (3×50 ml), dried over anhydrous Na₂SO₄, filtered and thesolvent removed under reduced pressure yielding 5.56 grams. The crudeproduct was dissolved in ethanol (60 ml) and glacial acetic acid (2.3grams). Girard's reagent T (5.6 grams) was added and the reactionmixture was refluxed for 2 hours. The reaction mixture was cooled inice-bath, alkaline solution (2.3 grams in 45 ml water) was added and themixture was extracted with ether (3×25 ml). The etheral phase was washedwith water and the water combined with the alkaline phase. The aqueousphase was acidified with concentrated HCl (4.4 ml) and extracted withether (3×25 ml). Washing with water, saturated aqueous sodiumbicarbonate (3×25 ml), water (2×25 ml), drying over anhydrous Na₂SO₄ andremoval of the solvent under reduced pressure gave 1.95 grams (0.0049mol) of 1-Hexadecyl-2-(5′-oxo-pentanyl)-glycerol (26.9% yield).

As depicted in Scheme IV, 1-Hexadecyl-2-(5′-oxopentyl)-glycerol (4.80grams) was dissolved in dry triethylamine (57 ml). Acetic anhydride (20ml) was added and the reaction mixture was stirred at room temperaturefor 2.5 hours. The reaction mixture was poured on ice (100 grams) andextracted with dichloromethane (3×100 ml). The organic phase was washedconsecutively with water (100 ml), diluted hydrochloric acid (100 ml),water (100 ml), saturated aqueous sodium bicarbonate (100 ml) and againwith water (100 ml) and was then dried over anhydrous sodium sulfate.The solvent removed under reduced pressure to give 4.54 grams of1-Hexadecyl-2-(5′-oxopentyl)-3-acetate glycerol (yield 86%).

1-Hexadecyl-2-(5′-oxopentyl)-3-acetate glycerol (3.94 grams) wasdissolved in t-butanol (75 ml). Sodium chlorite (6.85 grams) and sodiumdihydrogen phosphate dihydrate (15.50 grams) were dissolved in water (75ml). The aqueous solution was added to the alcoholic solution and thereaction mixture was e at room temperature for 4 hours. The reactionmixture was then transferred to separatory funnel and extracted withdichloromethane (3×100 ml). The combined organic phase was washed withwater (2×100 ml) and the solvent was removed under reduced pressure. Theresidue was dissolved in a mixture of methanol (80 ml) and 10% aqueousNaOH (20 ml) and the solution was stirred at room temperature overnight.The methanolic solution was extracted with a mixture of toluene andhexane (1:1) (2×50 ml), cooled in ice-bath and HCl conc. was addedslowly to reach pH 5-6. The solution was then extracted withdichloromethane (2×100 ml). The combined organic phase was washed withwater (100 ml), dried over anhydrous Na₂SO₄ and the solvent was removedunder reduced pressure to give 2.07 grams of a crude product.Recrystallization from hexane (20 ml) gave 1.30 grams of pure1-Hexadecyl-2-(4′-carboxy)butyl-glycerol (yield 35%).

As depicted in Scheme V, to the residue methanol (100 ml) and 10%aqueous NaOH (20 ml) were added and the resulting solution stirred atroom temperature for 2 hours. The solution was extracted with mixture ofpetroleum ether/toluene (1:1, v/v) and the methanolic phase acidified topH=0 with concentrated HCl and then extracted with chloroform (3×50 ml).The solvent was removed under reduced pressure and the residue wasdissolved in methanol (20 ml). Concentrated HCl (3 drops) was added andthe solution stirred at room temperature over night followed byextraction with chloroform (2×25 ml). The combined chloroform phase waswashed with water (2×50 ml), dried over anhydrous Na₂SO₄ and the solventremoved under reduced pressure yielding 0.77 gram (0.00179 mol) ofrac-1-Hexadecyl-2-(5′-pentanoic methyl ester)-glycerol (96.7% yield).

Example 2 Preparation of rac-1-hexadecyl-2-(5′-pentanoic methylester)-glycerol using periodate and an acetate protecting group

In this example, an unsaturated moiety is introduced into a glycerolicbackbone and is thereafter oxidized to an ester via an epoxide by meansof acetic anhydride, 4-chlorobenzoperoxoic acid, HClO₄ periodate andmethanol. Efficient isolation of the intermediates is performed bycarrying out the reactions while using an acetate protecting group

As a representative example, the preparation ofrac-1-hexadecyl-2-(5′-pentanoic methyl ester)-glycerol is herebydescribed.

rac-1-Hexadecyl-2-(5′-pentanoic methyl ester)-glycerol is prepared inaccordance with the teachings of the present invention, as is describedin Schemes VI through X below.

As depicted in Scheme VI below,1-Hexadecyl-2-(5′-hexenyl)-3-tritylglycerol, prepared as described inExample 1 above (4.90 grams) was dissolved in a mixture of methanol (30ml) and concentrated hydrochloric acid (3 ml) and the resulting solutionwas heated to reflux for 4 hours. The reaction mixture was cooled toroom temperature, poured on ice (100 grams) and extracted withchloroform (3×100 ml). The organic phase was washed with water (100 ml),aqueous sodium bicarbonate (100 ml) and again with water (100 ml).Thereafter the organic phase was dried over anhydrous Na₂SO₄, filteredand the solvent was removed to afford 3.75 grams of a residue. Theresidue was dissolved in n-hexane and kept at 4° C. overnight.Filtration of the precipitate and removal of the solvent gave 3.17grams, which were dissolved in chloroform (200 ml) and added to silicagel (45 grams). This solution was filtered and the silica gel extractedagain with mixture of chloroform:methanol (200 ml, 9:1) andchloroform:methanol (200 ml, 1:1). The two last extracts were combinedand the solvent removed under reduced pressure to afford 2.56 grams of1-Hexadecyl-2-(5′-hexenyl)-glycerol (84% yield).

As depicted in Scheme VII below, dry pyridine (5 ml) and aceticanhydride (3 ml) were added to the resulting1-Hexadecyl-2-(5′-hexenyl)-glycerol and the reaction mixture heated at70° C. for 2 hours. The reaction mixture was poured on ice (25 gram) andextracted with hexane (3×25 ml). The extract was washed successivelywith water (25 ml), aqueous diluted sulfuric acid (25 ml), water (25ml), aqueous sodium bicarbonate (25 ml) and water. After drying overanhydrous Na₂SO₄, filtration and removal of the solvent 2.60 grams wereobtained. The residue was dissolved in dichloromethane (50 ml) and3-chloroperbenzoic acid (3.84 grams) added, and the reaction mixture wasstirred at room temperature for over night. The solvent was reduced toabout 20 ml under reduced pressure and n-hexane (100 ml) was added.After filtration the solvent was evaporated to dryness. The residue wasdissolved in n-hexane (100 ml), alkaline solution (0.4 grams NaOH in 50ml of water) was added and the phases were separated. Washing of theorganic phase successively with water (25 ml), aqueous sodiumbicarbonate (25 ml), water (25 ml), drying over anhydrous Na₂SO₄,filtration and removal of the solvent afforded 2.40 grams of1-Hexadecyl-2-(5′,6′-epoxyhexanyl)-3-acetate glycerol (82% yield).

As depicted in Scheme VIII below,1-Hexadecyl-2-(5′,6′-epoxyhexanyl)-3-acetate glycerol was dissolved inacetone (50 ml). 7% HClO₄ (5 ml) was added and the reaction mixturestirred at room temperature for 40 hours. Water (50 ml) was added andthe reaction mixture extracted with chloroform (3×50 ml). Washing of theorganic phase successively with water (25 ml), aqueous sodiumbicarbonate (25 ml), water (25 ml), drying over anhydrous Na₂SO₄,filtration and removal of the solvent gave 2.29 grams of oily residue.The residue was dissolved in chloroform (200 ml) and added to silica gel(30 grams). This solution was filtered and the silica gel extractedagain with mixture of chloroform:methanol (200 ml, 8:2). In the secondextract after solvent removed under reduced pressure 1.45 grams of1-Hexadecyl-2-(5′,6′-dihydroxyhexanyl)-3-acetate glycerol were obtained.

As depicted in Scheme IX below,1-Hexadecyl-2-(5′,6′-dihydroxyhexanyl)-3-acetate glycerol was dissolvedin isopropanol (50 ml). Aqueous solution of sodium periodate (1.45 gramsin 50 ml of water) was added and the reaction mixture stirred at roomtemperature for 2 hours. The reaction mixture was extracted withchloroform (3×50 ml), dried over anhydrous Na₂SO₄, filtered and thesolvent removed under reduced pressure yielding 0.96 gram. The residuewas dissolved in t-butanol (50 ml) and aqueous solution (50 ml) ofsodium chlorite (1.66 grams) and sodium dihydrogen phosphate dihydrate(3.76 grams) was added. The reaction mixture was stirred at roomtemperature for 4 hours, extracted with chloroform (2×50 ml) and thesolvent removed under reduced pressure. The residue was dissolved inmixture of chloroform:hexane (200 ml, 1:1) and added to silica gel (15grams). The solution was filtered and the silica gel extracted againwith chloroform (200 ml) and chloroform:methanol (200 ml, 9:1). Thesolvent from the last extract was removed under reduced pressure to give0.92 grams of 1-Hexadecyl-2-(4′-carboxybutyl)-3-acetate glycerol.

As depicted in Scheme X below, 1-Hexadecyl-2-(4′-carboxybutyl)-3-acetateglycerol dissolved in 50 ml of an 8:2 mixture of methanol and 10%aqueous NaOH and the reaction mixture is stirred vigorously at roomtemperature overnight. The reaction mixture is extracted with mixture oftoluene:petroleum ether (2×25 ml, 1:1). The methanolic phase isacidified with concentrated HCl until reaching pH of about 0, and thenextracted with chloroform (2×25 ml). The solvent was removed underreduced pressure and the residue was dissolved in methanol (10 ml).Concentrated HCl (2 drops) is added and the solution is stirred at roomtemperature over night followed by extraction with chloroform (2×25 ml),successive washing of the organic phase with water (25 ml), followed bywashing with aqueous sodium bicarbonate (25 ml), water (25 ml), followedby drying over anhydrous Na₂SO₄, filtration and removal of the solventto afford 0.86 grams of pure rac-1-Hexadecyl-2-(5′-pentanoic methylester)-glycerol.

Example 3 Preparation of rac-1-hexadecyl-2-(5′-pentanoic ethylester)-glycerol by direct introduction of an oxidized moiety —Route I

rac-1-Hexadecyl-2-(5′-pentanoic ethyl ester)-glycerol is prepared inaccordance with the teachings of the present invention, as is describedin Scheme XI below.

1-Hexadecyl-3-tritylglycerol is prepared as described, for example, inExample 1 above or as described in U.S. Pat. No. 6,838,425.

To a three-necked flask equipped with a magnetic stirrer, 1.0 gram (1.8mmole) 1-hexadecyl-3-tritylglycerol, 0.78 gram (3.6 mmole)5-bromovaleric acid ethyl ester and 75 ml dimethylformamide (DMF) areadded. To the stirred solution, 0.20 gram (5 mmole) NaH (60% dispersionin mineral oil) dissolved in 25 ml dimethylformamide are added dropwiseover 15 minutes and stirring is continued for an additional 1 hour untilthe reaction is completed. Water was added (50 ml) and the mixtureextracted with ether (3×50 ml). The organic phase was dried overanhydrous Na₂SO₄ and the solvent removed under reduced pressure. Thecrude product was purified over column chromatography on silica gel.

Deprotection of the trityl group as described hereinabove gave the finalproduct.

Example 4

Introduction of a phosphorous-containing moiety to glycerolipid compound

According to the teachings of the present invention, a reactivephosphorous-containing moiety is introduced into a glycerolipid compoundhaving one or two oxidized (or pre-oxidized) moiety-containing residuesattached thereto via an ether bond. The introduction of the reactivephosphorous-containing moiety is performed using aphosphorous-containing compound such as, for example, phosphorousoxychloride). Optionally, subsequent to the introduction of the reactivephosphorous-containing moiety, the reactive phosphorous-containingmoiety is converted to a phosphate moiety.

Preparation of rac-1-hexadecyl-2-(5′-hexenyl)-3-dichlorophosphate:

As a representative example,rac-1-Hexadecyl-2-(5′-hexenyl)-3-dichlorophosphate was prepared inaccordance with the teachings of the present invention, as is describedin scheme XII below.

Thus, 0.24 ml (0.39 gram, 2.53 mmole) POCl₃ and 10 ml tetrahydrofuran(THF) are placed in an ice-cooled three-necked flask equipped with amagnetic stirrer. To the stirred solution was added dropwise, over 25minutes, a mixture of 0.87 gram (2.2 mmole)rac-1-Hexadecyl-2-(5′-hexenyl)-glycerol, 0.34 ml (0.25 gram, 2.44 mmole)triethylamine and 50 ml tetrahydrofuran (THF) and stirring is continuedfor an additional 10 minutes in an ice-bath and further continued for 45minutes at 23° C.

The rac-1-Hexadecyl-2-(5′-hexenyl)-3-dichlorophosphate can behydrolyzed, to thereby produce the corresponding phosphatidic acid, asfollows:

One gram of ice is added to the reaction mixture and stirring iscontinued for 30 minutes. Water (50 ml) is then added and the product isextracted with mixture of chloroform:MeOH (2:1, v/v, 3×25 ml). Theorganic phase is washed with water and the solvent removed under reducedpressure.

Alternatively, the rac-1-Hexadecyl-2-(5′-hexenyl)-3-dichlorophosphatecan be reacted with various alkylamine derivatives, to thereby produce aphosphoglyceride, as is exemplified below.

Preparation of rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphoethanolamine fromrac-1-Hexadecyl-2-(5′-hexenyl)-3-dichlorophosphate:

rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphoethanolamine was prepared inaccordance with the teachings of the present invention, as is describedin scheme XIII

A solution of rac-1-hexadecyl-2-(5′-hexenyl)-3-dichlorophosphate in THFprepared as described immediately hereinabove in Example 2 was cooled inan ice bath. To the solution was added dropwise over a period of 10minutes a mixture of 0.16 ml (0.16 gram, 2.7 mmole) ethanolamine, 0.34ml (0.25 gram, 2.4 mmole) triethylamine and 50 ml THF. After all thesolution was added, the resulting solution was stirred for an additional20 minutes and then removed from the ice bath and stirred overnight atroom temperature.

The solution was filtered using filter paper (Whatman #2). The residueremaining on the filter paper was dried under reduced pressure to yield1.2 gram of an off-white residue.

The 1.2 gram off-white residue was dissolved in a mixture of 24 mlglacial acetic acid and 10 ml water, maintained at 70° C. for 1 hour andallowed to cool to room temperature. The product was extracted from theacetic acid solution by twice washing with 50 ml of a 2:1chloroform:methanol extraction solution. The solvents of the extractionsolution were evaporated leaving 0.94 gram (1.7 mmol)rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphoethanolamine, a yield of 85%relative to the rac-1-Hexadecyl-2-(5′-hexenyl)-glycerol.

Preparation of rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphocholine fromrac-1-hexadecyl-2-(5% hexenyl)-3-phosphoethanolamine

rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphocholine was prepared inaccordance with the teachings of the present invention, as is describedin scheme XIV below.

To a three-necked flask equipped with a magnetic stirrer 0.50 gram (0.99mmole) rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphoethanolamine, 50 mlisopropanol and 18 ml CH₂Cl₂ are added. While stirring, a mixture of 5gram K₂CO₃ and 10 ml water was added and the temperature of the solutionwas maintained at between about 35° C. and about 40° C. while a mixtureof 1.0 ml (1.3 gram, 11 mmole) dimethylsulfate and 10 ml isopropanol wasadded dropwise over a period of 45 minutes. After all the solution wasadded, the solution was stirred for an additional 90 minutes. Thesolution was allowed to cool to room temperature. The resulting productwas extracted from the solution by thrice washing with 50 ml of a 2:1chloroform:methanol solution. The solvents of the solution wereevaporated leaving 0.50 gram (0.82 mmole)rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphocholine, a yield of 92% yieldrelative to rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphoethanolamine.

Purity was confirmed with thin-layer chromatography on alumina using anelution solvent of chloroform:methanol:water (70:26:4). The identity ofthe rac-1-hexadecyl-2-(5′-hexenyl)-3-phosphocholine was confirmed using¹³C-NMR.

Example 5 Preparation of1-Hexadecyl-2-(4′-carboxymethyl)butyl-3-phosphocholine

A solution of 1-Hexadecyl-2-(5′-carboxymethyl)butyl-glycerol (0.86grams), 0.34 gram (2.6 mmole) triethylamine and 50 ml tetrahydrofuranwas added dropwise, over 25 minutes to an ice-cooled solution of 0.24 ml(0.39 gram, 2.6 mmole) POCl₃ and 10 ml tetrahydrofuran (THF). Theresulting mixture was stirred for additional 10 minutes in an ice-bathand for 45 minutes at room temperature (23° C.). The reaction mixturewas then cooled in an ice-bath and a solution of ethanolamine (0.16 ml)and triethylamine (0.64 ml) in THF (50 ml) was added dropwise theretounder vigorous stirring. The stirring was continued for additional 10minutes in an ice-bath and further continued at room temperature forovernight. The reaction mixture was then filtered and the solventremoved under reduced pressure. The residue was dissolved in a mixtureof acetic acid (24 ml) and water (10 ml) and the solution was heated to70° C. for 1 hour. After cooling to room temperature, the mixture wasextracted with chloroform (2×25 ml) and the solvent was removed underreduced pressure. The residue was dissolved in a mixture of iso-propanol(50 ml) and dichloromethane (18 ml). Potassium carbonate (5.0 gram) inwater (10 ml) was added thereto and the resulting mixture was warmed to35-40° C. A solution of dimethylsulfate (1 ml) in 10 ml iso-propanol wasthen added dropwise over 45 minutes. After additional 90 minutes themixture was extracted with chloroform (3×50 ml) and the solvent wasremoved under reduced pressure to give 1.10 grams of1-Hexadecyl-2-(4′-carboxymethyl)butyl-3-phosphocholine (92% yield).

Preparation of 1-Hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine:1-Hexadecyl-2-(4′-carboxymethyl)butyl-3-phosphocholine was dissolved inmethanol (25 ml). Sodium hydroxide (1.0 gram) dissolved in 90% methanol(20 ml) was added to the methanolic solution and the reaction mixturewas stirred at room temperature for 5 hours. The pH of the reaction wasadjusted to 4 by adding sodium dihydrogen phosphate. Water (50 ml) andchloroform (50 ml) were added, the organic phase was collected and thesolvent was removed under reduced pressure. The residue was dissolved inchloroform, dried over anhydrous Na₂SO₄, filtered and the solvent wasremoved under reduced pressure.1-Hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine (0.71 grams) wereobtained (66% yield).

Example 6 Preparation of1-hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine (CI-201) via directoxidation of an unsaturated bond (a scalable process)

A process of preparing 1-hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine(CI-201), which can be readily scaled-up for industrial manufacturing ofthe product is depicted in Scheme XV below:

In this process, 1-hexadecyl-2-(5′-hexenyl)-3-tritylglycerol is directlyoxidized to obtain the corresponding carboxylic acid in a one-stepprocedure, thus circumventing the need to perform the oxidation via amultiple-step procedure that requires laborious separations of theintermediates. The oxidation step is performed using safe, efficient andless hazardous oxidizing agents. Purification procedures of all theintermediates are performed while avoiding the use of industriallyinapplicable column chromatography.

This process was efficiently scaled-up, so as to industriallymanufacture CI-201.

Preparation of 1-Hexadecyl-glycerol:(R)-(−)-2,2-dimethyl-1,3-dioxolane-4-methanol (11 grams), powderedpotassium hydroxide (20 grams) and hexadecyl bromide (27.96 grams) intoluene (150 ml) were stirred and refluxed for 6 hours, while removingthe water formed by azeotropic distillation. The volume of the solventwas gradually reduced to about 40 ml. The reaction mixture was cooled toroom temperature; water was added (100 ml) and the resulting mixture wasextracted with dichloromethane (3×75 ml). The combined organic phase waswashed with water (50 ml) and the solvent removed under reducedpressure. The residue was dissolved in 200 ml mixture of 90:10:5methanol:water:concentrated hydrochloric acid (v/v) and the resultingsolution was refluxed for 2 hours, followed by cooling to roomtemperature and addition of water (100 ml). The product was extractedwith dichloromethane (3×100 ml), and the organic phase was washedconsecutively with water (100 ml), saturated aqueous solution of sodiumcarbonate (100 ml) and again with water (100 ml). The solvent wasremoved under reduced pressure and the product was crystallized fromhexane (200 ml) to give 21.69 grams (yield 82%) of pure1-hexadecyl-glycerol, upon drying in a desiccator under reducedpressure.

Preparation of 1-Hexadecyl-3-trityl-glycerol: 1-Hexadecyloxy-glycerol(20 grams) and triphenylchloromethane (21.29 grams) were placed in dryTHF (369 ml) and dry acetonitrile (93 ml). Triethylamine (17.75 ml) wasadded and the reaction mixture was refluxed for 17 hours. The reactionmixture was thereafter cooled to room temperature, poured on ice (100grams), transferred to a separatory funnel and extracted with ether. Theorganic phase was washed consecutively with water (200 ml), diluted(1.5%) H₂SO₄ (2×200 ml), water (200 ml), saturated aqueous sodiumbicarbonate (200 ml) and again with water (200 ml), dried over anhydroussodium sulfate and the solvent removed under reduced pressure to give36.86 grams of crude product.

The residue was dissolved in hot hexane (200 ml) and the resultingsolution was cooled at 4° C. overnight. The resulting precipitate wasfiltered to yield 23.77 grams of the purified compound. Additionalpurified product was collected by removing the solvent from the motherliquor under reduced pressure and dissolving the residue again in hothexane (50 ml). The resulting solution was cooled at 4° C. overnight andthe precipitate filtered to afford additional 6.94 grams of the productand a total amount of 30.71 grams.

Preparation of 1-Hexadecyl-2-(5′-hexenyl)-3-tritylglycerol:1-Hexadecyl-3-tritylglycerol (19.94 grams), 6-bromo-1-hexene (6.98grams, 5.73 ml) and powdered potassium hydroxide (15 grams) in hexane(350 ml) were stirred and refluxed for 8 hours, while removing the waterformed by azeotropic distillation. The reaction mixture was then cooledto room temperature, transferred to a separatory funnel and washed withwater (2×200 ml). The solvent was thereafter removed under reducedpressure and the residue was dissolved in hexane (150 ml) and washedagain with water (2×200 ml). The organic solution was kept at 4° C.overnight, during which precipitation of byproducts occurred. Filtrationand removal of the solvent under reduced pressure gave 19.86 grams(86.6% yield) of 1-hexadecyl-2-(5′-hexenyl)-3-tritylglycerol.

Preparation of 1-hexadecyl-2-(4′-carboxy)butyl-sn-glycerol: In athree-neck round bottom flask equipped with thermometer and droppingfunnel, sodium periodate (150.16 grams, 702 mmol, 9 equivalents) weresuspended in 500 ml water. After addition of sodium bicarbonate (7.21grams, 85.8 mmol, 1.1 equivalents) and potassium permanganate (2.47grams, 15.6 mmol, 0.2 equivalent), the suspension was heated to 40° C.1-Hexadecyl-2-(5′-hexenyl)-3-tritylglycerol (50.00 grams, 78.0 mmol) wasdissolved in tert-butanol (500 ml) and the solution was added to theNaIO₄/KMnO₄ mixture during 1 hour. After 1.5 hours, analysis by TLCshowed 80% conversion. Additional amount of potassium permanganate (0.62gram, 3.9 mmol, 0.05 equivalent) was added and the mixture was stirredfor 1.5 hours. Analysis by TLC showed less than 5% of the startingmaterial. The reaction mixture was then cooled to room temperature andtransferred to separation funnel.

The intermediate 1-Hexadecyl-2-(4′-carboxy)butyl-3-tritylglycerol wasextracted with hexane (200 ml). The organic phase was washed with asolution of Na₂S₂O₅ (15 grams) in 100 ml water. Diluted hydrochloricacid (0.65 ml concentrated HCl in 13 ml water) was added to the organicphase and 200 ml of the solvent were distilled under reduced pressure.The remaining clear solution was heated to 80° C. for 6 hours. Analysisby TLC showed less than 5% of intermediate1-Hexadecyl-2-(4′-carboxy)butyl-3-tritylglycerol. Additional volume of250 ml solvent was distilled off.

The residue was treated with 100 ml water and 10 ml 30% NaOH to reachpH=12. The precipitated triphenylmethanol was filter off and washed 4times with 10 ml water. The filtrate was extracted with a mixture of 50ml hexane and 50 ml ethyl acetate to remove remaining triphenylmethanoland other impurities. The sodium salt of1-hexadecyl-2-(4′-carboxy)butyl-sn-glycerol, present in the aqueousphase, was protonated with concentrated hydrochloric acid (8.45 ml,101.4 mmol, 1.3 equivalents, pH=1). The resulting free carboxylic acidwas extracted with hexane (100 ml). Evaporation to dryness andco-evaporation with 100 ml hexane gave 27.00 grams of crude1-hexadecyl-2-(4′-carboxy)butyl-sn-glycerol.

The crude product was crystallized by dissolving in a mixture of acetoneand hexane (7 ml/68 ml) and cooling to 0° C. The precipitate wasfiltered and washed with cold hexane (2×7 ml) and dried.1-Hexadecyl-2-(4′-carboxy)butyl-sn-glycerol was obtained as an off-whitesolid (20.90 grams, 50.2 mmol, 64.3% yield).

Preparation of 1-Hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycerol:1-Hexadecyl-2-(4′-carboxy)butyl-sn-glycerol (15.0 grams, 36.0 mmol) wasdissolved in methanol (100 ml) and concentrated hydrochloric acid (3 ml)was added. The reaction mixture was stirred at room temperatureovernight. Triethylamine was thereafter added until the reaction mixturereaches pH=7. The solution was transferred to separatory funnel andextracted with hexane (2×200 ml). The organic phase was washed withwater and evaporation to dryness and co-evaporation with 100 ml hexanegave 14.92 grams of 1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycerol(34.65 mmol, 96.2% yield).

1-Hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphocholine: Asolution of 1-Hexadecyl-2-(4′-carboxymethyl)butyl-glycerol (8.60 grams,19.97 mmol), and triethylamine (2.63 grams, 3.62 ml, 26 mmol) in 500 mlTHF was added dropwise, over 25 minutes, to an ice-cooled solution ofPOCl₃ (3.90 grams, 2.40 ml, 26 mmol) in 100 ml THF. The resultingmixture was stirred for an additional 10 minutes in an ice-bath and for45 minutes at room temperature (23° C.). A solution of ethanolamine (1.6ml) and triethylamine (6.4 ml) in THF (500 ml) was then added dropwiseunder vigorous stirring to an ice-cooled reaction mixture. The stirringwas continued for an additional 10 minutes in an ice-bath and furthercontinued at room temperature for overnight. The reaction mixture wasthereafter filtered and the solvent removed under reduced pressure. Theresidue was dissolved in a mixture of acetic acid (24 ml) and water (100ml) and heated to 70° C. for 1 hour. The reaction mixture was thereaftercooled to room temperature and extracted with dichloromethane (2×250ml). The solvent was removed under reduced pressure, to afford crude1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoethanolamine.

The crude1-hexadecyl-2-(4′-carboxymethyl)butyl-sn-glycero-3-phosphoethanolaminewas dissolved in a mixture of isopropanol (500 ml) and dichloromethane(180 ml). A solution of potassium carbonate (50 grams) in water (100 ml)was added to reach a pH above 11, and the solution was kept at 35-40° C.during the dropwise addition of methyltosylate (11.15 grams) in 100 mlof iso-propanol in a time period of 45 minutes. After additional 90minutes, the mixture was acidified with hydrochloric acid. Water (100ml) and dichloromethane (550 ml) were added and the phases separated.The organic phase was washed with water (100 ml) and the solvent removedunder reduced pressure to give 11.0 grams of1-hexadecyl-2-(5′-carboxymethyl)butyl-3-phosphocholine (18.46 mmol,92.45% yield).

Preparation of 1-Hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine:1-Hexadecyl-2-(4′-carboxymethyl)butyl-3-phosphocholine was dissolved inisopropanol (250 ml). Lithium hydroxide monohydrate (1.68 grams) wasadded and the reaction mixture was stirred at room temperatureovernight. Isopropanol was partially evaporated by distillation and thepH of the reaction was brought acidic by addition of hydrochloric acid.Water (250 ml) was added and the solution extracted with dichloromethane(2×250 ml). The solvent was thereafter removed under reduced pressureand co-evaporated with dichloromethane to give crude1-hexadecyl-2-(5′-carboxy)butyl-3-phosphocholine.

The crude 1-hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine was purifiedby chromatography on a silica gel column. Dichloromethane followed by amixture of dichloromethane, methanol, water, and triethylamine was usedto elute the product from the column. The fractions containing theproduct were combined and evaporated. The resulting product was driedunder vacuum. 7.10 grams of pure1-hexadecyl-2-(4′-carboxy)butyl-3-phosphocholine (12.2 mmol, 66.1%yield) were obtained.

Example 7 Preparation of1-hexadecyl-2-(6′-carboxy)hexanyl-sn-glycero-3-phosphocholine directintroduction of oxidized moiety—Route II

Preparation of1-hexadecyl-2-(6′-carboxyethyl)hexanyl-3-trityl-sn-glycerol:

To a solution of 1-hexadecyl-3-trityl-sn-glycerol (5.0 grams, 8.95mmol), and ethyl 7-bromo-heptanoate (2 ml, 2.44 grams, 10.29 mmol) inbenzene (70 ml), powdered KOH (23 grams) was added. The reaction mixturewas stirred and refluxed for 14 hours, while removing the water formedby azeotropic distillation. The reaction mixture was cooled to roomtemperature, washed with water (3×70 ml) and dried over anhydrous sodiumsulfate. The solvent was removed under reduced pressure and the residuewas dissolved in hexane (25 ml) and cooled to 4° C. The byproductprecipitated and was filtered off. The solvent was removed from thefiltrate under reduced pressure to give 5 grams of1-hexadecyl-2-(6′-carboxyethyl)hexanyl-3-trityl-sn-glycerol as a whitesolid (6.99 mmol, 78.1% yield).

Preparation of 1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycerol:

To a solution of1-hexadecyl-2-(6′-carboxyethyl)hexanyl-3-trityl-sn-glycerol (5.0 grams,7 mmol) in ethanol (90 ml), concentrated hydrochloric acid (32%, 20 ml)was added slowly. The reaction mixture was stirred and refluxed for 4hours and was thereafter cooled to room temperature, poured on ice andextracted with diethyl ether (3×100 ml). The organic phase was washedwith water (100 ml), saturated aqueous sodium bicarbonate solution (100ml) and water (100 ml). After drying over anhydrous sodium sulfate andfiltration, the solvent was removed under reduced pressure. N-hexane wasadded and the mixture kept at 4° C. overnight. After filtration of theprecipitate, the filtrate was concentrated by evaporation and the yellowsolution was kept at 4° C. overnight. After filtration of theprecipitate the yellow solution was warmed to room temperature and thesolvent removed under reduced pressure to give 3.1 grams yellow oil. Theresidue was purified by chromatography on silica gel column (140 grams).The elution started with 300 ml chloroform, the polarity increased to300 ml CHCl₃: EtOAc 90%:10% then to 300 ml CHCl₃: EtOAc 80%:20% and 300ml CHCl₃: EtOAc 70%:30% and finally to 300 ml CHCl₃: EtOAc 60%:40%. Theproduct was collected from fractions eluted by the latter two eluentmixtures, upon combining the fractions and removing the solvent underreduced pressure. 1.34 grams of colorless oil were obtained, and driedunder reduced pressure with phosphorus pentoxide to give1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycerol as a colorless solid(2.83 mmol, 40.5% yield).

Preparation of1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycero-3-phosphoethanolamine:

1-Hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycerol (1.34 gram, 2.83mmol) and triethylamine (1.2 ml) were dissolved in 15 ml THF. Thesolution was added dropwise for over 15 minutes to an ice-cooledsolution of POCl₃ (0.8 ml, 8.5 mmol) in 10 ml THF. The stirring wascontinued for additional 10 minutes with cooling and further continuedfor 45 minutes at room temperature. A solution of ethanolamine (0.52 ml,8.5 mmol) and triethylamine (2.4 ml) in THF (25 ml) was added dropwiseover 15 minutes to the ice-cooled reaction mixture. The stirring wascontinued for 10 minutes, the cooling bath was thereafter removed andthe reaction mixture was stirred at room temperature overnight. Thereaction mixture was then filtered and the solvent removed under reducedpressure. The residue was dissolved in a mixture of acetic acid (24 ml)and water (10 ml) and heated to 70° C. for 1 hour. The mixture wasthereafter extracted with chloroform (3×50 ml), the organic phase washedwith water (2×50 ml) and the solvent removed under reduced pressure togive 1.87 grams of crude1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycero-3-phosphoethanolamineas yellow oil.

Preparation of1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycero-3-phosphocholine:

1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycero-3-phosphoethanolaminewas dissolved in mixture of isopropanol (50 ml) and dichloromethane (18ml). A solution of potassium carbonate (2.17 grams) in water (10 ml) wasadded dropwise over 5 minutes while keeping the solution at 35-40° C.Dimethylsulfate (1.52 ml, 15.69 mmol) in isopropanol (10 ml) was addeddropwise at 40° C. during 10 minutes and the reaction was stirred at 40°C. for 90 minutes. Water was then added and the mixture was extractedwith chloroform (2×50 ml). The organic phase was washed with water (50ml) and the solvent was removed under reduced pressure to give 1.8 gramsof 1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycero-3-phosphocholine aswax.

Preparation of1-hexadecyl-2-(6′-carboxy)hexanyl-sn-glycero-3-phosphocholine:

To mixture of1-hexadecyl-2-(6′-carboxyethyl)hexanyl-sn-glycero-3-phosphocholine (1.8grams, 2.82 mmol) in methanol (50 ml), a solution of 10% sodiumhydroxide was added. The mixture was stirred at room temperature for 5hours. The pH of the reaction was adjusted to 4-5 by adding sodiumdihydrogen phosphate, and water (70 ml) and chloroform (70 ml) wereadded. The phases were separated and the solvent was removed underreduced pressure. The residue was dissolved in chloroform, dried overanhydrous sodium sulfate, filtered and the solvent removed under reducedpressure to give 1.29 grams of the crude product as a white wax.

The crude product was purified by chromatography on silica gel column(62 grams). The elution started with 200 ml CHCl₃: MeOH 80%:20% to elutethe non polar residues, then the polarity increased to 200 mlCHCl₃:MeOH:Water 70%:26%: 4% and finally to 300 ml CHCl₃:MeOH:Water60%:35%:5%. The product was eluted with the final mixture. The fractionswere collected, the solvent was removed under reduced pressure, theresidue was dissolved in chloroform and dried over anhydrous sodiumsulfate and the solvent was removed reduced pressure. The product wasdried under reduced pressure with phosphorus pentoxide. 1.0 gram of1-hexadecyl-2-(6′-carboxy)hexanyl-sn-glycero-3-phosphocholine as whitewax was obtained (58.1% yield).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A method of preparing a compound having aglycerolic backbone and at least one oxidized moiety-containing residueattached to the glycerolic backbone via an ether bond, the compoundhaving the general Formula II:

wherein: A₁ is selected from the group consisting of CH₂, CH═CH and C═O;A₂ is C₂:, R₁ is an alkyl having 1-30 carbon atoms; R₂ is

whereas: X is an alkyl chain having 1-24 carbon atoms; Y is hydrogen;and Z is

R₃ is selected from the group consisting of hydrogen, alkyl, aryl,phosphoric acid, phosphoryl choline, phosphoryl ethanolamine, phosphorylserine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylserine, phosphatidyl cardiolipin, phosphatidyl inositol, phosphorylcardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, pho sphorylethanolamine-N-lacto se,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinosito1-4,5-biphosphonate,pyrophosphate, pho sphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, and phosphoglycerol, the methodcomprising: providing a first compound having a glycerolic backbone andat least one free hydroxyl group, said first compound having the generalformula I:

wherein: A₁ is absent or is selected from the group consisting of CH₂,CH═CH and C═O: R₁ is selected from the group consisting of H and ahydrocarbon chain having from 1 to 30 carbon atoms; and R₃ is selectedfrom the group consisting of hydrogen, alkyl, aryl, phosphoric acid,phosphoryl choline, phosphoryl ethanolamine, phosphoryl serine,phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine,phosphatidyl cardiolipin, phosphatidyl inositol, phosphoryl cardiolipin,phosphoryl inositol, ethylphosphocholine, phosphorylmethanol,phosphorylethanol, phosphorylpropanol, phosphorylbutanol, phosphorylethanolamine-N-lacto se, phosphoethanolamine-N-[methoxy(propyleneglycol)], phosphoinositol-4-phosphate,phosphoinosito1-4,5-biphosphonate, pyrophosphate,phosphoethanolamine-diethylenetriamine -pentaacetate,dinitrophenyl-phosphoethanolamine, and phosphoglycerol; providing acompound having at least one oxidized moiety and at least one reactivegroup, said oxidized moiety being a carboxylic ester; reacting saidfirst compound and said compound having at least one oxidized moiety andat least one reactive group in the presence of an inorganic base, tothereby obtain a reaction mixture containing a compound having aglycerolic backbone and at least one carboxylic ester-containing residueattached to the glycerolic backbone via an ether bond, wherein when saidcompound having said at least one oxidized moiety and dais at least onereactive group is 5 carbon atoms in length, said reacting in thepresence of said inorganic base is performed under conditions which donot adversely affect reaction efficiency, and wherein when R₃ of saidcompound is hydrogen, the method further comprises, prior to saidreacting said first compound and said compound having said at least oneoxidized moiety and said at least one reactive group, protecting a freehydroxyl group at position sn-3 of said glycerolic backbone with aprotecting group; hydrolyzing said carboxylic ester so as to producesaid Z; and isolating the compound having a glycerolic backbone and atleast one oxidized moiety-containing residue attached to the glycerolicbackbone via an ether bond.
 2. The method of claim 1, wherein saidinorganic base is selected from the group consisting of sodium hydride,lithium aluminum hydride, sodium amide, sodium hydroxide, potassiumhydroxide and any mixture thereof.
 3. The method of claim 1, wherein thecompound having a glycerolic backbone and at least one oxidizedmoiety-containing residue attached to the glycerolic backbone furthercomprises a phosphorus-containing moiety attached to the glycerolicbackbone, such that R₃ is selected from the group consisting ofphosphoric acid, phosphoryl choline, phosphoryl ethanolamine,ephosphoryl serine, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl serine, phosphatidyl cardiolipin, phosphatidyl inositol,phosphoryl cardiolipin, phosphoryl inositol, ethylphosphocholine,phosphorylmethanol, phosphorylethanol, phosphorylpropanol,phosphorylbutanol, phosphorylethanolamine-N-lactose,phosphoethanolamine-N-[methoxy(propylene glycol)],phosphoinositol-4-phosphate, phosphoinositol-4,5-biphosphonate,pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,dinitrophenyl-phosphoethanolamine, and phosphoglycerol, the methodfurther comprising, prior to or subsequent to reacting said firstcompound and said compound having said at least one oxidized moiety andsaid at least one reactive group, or subsequent to isolating saidcompound having said at least one ester-containing residue attached tosaid glycerolic backbone: reacting said first compound or said compoundhaving said at least one ester-containing residue attached to saidglycerolic backbone with a phosphorus-containing moiety, to therebyobtain said compound having a glycerolic backbone and at least oneoxidized moiety-containing residue attached to the glycerolic backboneand further having a phosphorus-containing moiety attached to theglycerolic backbone.
 4. The method of claim 3, wherein reacting saidfirst compound or said compound having said at least oneester-containing residue attached to said glycerolic backbone with saidphosphorus-containing moiety comprises: providing said first compound orsaid compound having said at least one carboxylic ester-containingresidue attached to said glycerolic backbone, and further having a freehydroxyl group; reacting said first compound or said compound havingsaid at least one ester-containing residue attached to said glycerolicbackbone with a reactive phosphorus-containing compound having a secondreactive group capable of reacting with said free hydroxyl group and athird reactive group, to thereby provide said first compound or saidcompound having said at least one ester-containing residue attached tosaid glycerolic backbone, having a reactive phosphorus-containing groupattached thereto; and converting said reactive phosphorus-containingroup to said phosphorus-containing moiety.
 5. The method of claim 4,wherein said reactive phosphorus-containing compound is phosphorusoxychloride (POCl3).
 6. A method of introducing a phosphate moiety intoa compound having a glycerolic backbone and having an oxidizedmoiety-containing or a pre-oxidized moiety-containing residue attachedthereto via an ether bond, the method comprising: providing a compoundhaving a glycerolic backbone and an oxidized moiety -or a pre-oxidizedmoiety-containing residue attached to said glycerolic backbone via anether bond and at least one a free hydroxyl group; reacting saidcompound with a phosphorus-containing compound which is POCl₃, tothereby provide a compound having an oxidized moiety- or a pre-oxidizedmoiety-containing residue and a reactive phosphorus-containing group;and converting said reactive phosphorus-containing group to saidphosphate moiety, thereby introducing the phosphate moiety into thecompound.
 7. The method of claim 6, wherein said oxidized moiety isselected from the group consisting of carboxylic acid, ester, acylhalide, aldehyde, acetal, diol and ketal.
 8. The method of claim 6,wherein said pre-oxidized moiety is an unsaturated moiety.
 9. The methodof claim 1, wherein said compound having said at least one oxidizedmoiety and said at least one reactive group is more than 5 carbon atomsin length.
 10. The method of claim 9, wherein said compound having saidat least one oxidized moiety and said at least one reactive group isethyl 7-bromoheptanoate.